Supports having azlactone-functional surfaces, adduct supports, and methods of preparing both

ABSTRACT

Supports having azlactone-functional surfaces, adduct supports prepared from such azlactone-functional supports, and methods of preparing both are disclosed. Azlactone functionality is introduced to surfaces of a pre-existing support in a manner which retains useful physical and chemical characteristics of the pre-existing support. One method involves exposing surfaces with high energy radiation to generate free radical reaction sites on the surfaces and causing azlactone-functional moieties to react with the free radical reaction sites. Another method involves coating surfaces with azlactone monomers, crosslinking monomers, and optionally co-monomers and polymerizing the monomers to form a polymerized coating of azlactone-functionality on the surfaces. Another method involves dispersion polymerization of azlactone-functional moieties to produce azlactone-functional particles within pores and interstices of a pre-existing support. Adduct supports are formed by coupling nucleophilic reagents, such as biologically active materials, to azlactone-functional moieties of the support.

This is a division of application Ser. No. 07/896,107 filed Jun. 9, 1992now U.S. Pat. No. 5,344,701.

FIELD OF THE INVENTION

This patent application relates to supports having azlactone-functionalsurfaces, adduct supports prepared from such azlactone-functionalsupports, and methods of preparing both.

BACKGROUND OF THE INVENTION

Azlactone-functional polymeric supports have been prepared according tothe methods disclosed in European Patent Publication 0 392 783 (Colemanet al.) and in European Patent Publication 0 392 735 (Heilmann et al.).In both of these publications, examples show methods of preparationwhich involve the homopolymerization or copolymerization ofazlactone-functional polymers to become the polymeric support.

Azlactone-functional moieties are expensive and valuable. Preparationtechniques which cause azlactone-functional moieties to be occluded fromaccessible use needlessly wastes the valuable azlactone functionality.

Also, there is a desire to place azlactone-functionality only atsurfaces of a support where chemical or physical interaction with othermaterials, particularly biologically active materials can occur. ProcessIV described in European Patent Publication 0 392 735 discloses a methodfor coating azlactone-containing polymer at surfaces of substrates.Also, European Patent Publication 0 392 735 within the disclosure ofProcess IV and in Example 22 thereof identifies a desire to employazlactone-containing monomers in the coating process to polymerize themonomer(s) in place.

There are a myriad of supports which have specific geometries useful forphysical interaction with materials, particularly biologically activematerials. These supports have specific physical and chemicalcharacteristics: porosity, surface area, permeability, solventresistance, hydrophilicity, flexibility, mechanical integrity, and otherstability or feature in the use environment, etc., which must beretained for a pre-existing support to remain useful. For example, amicroporous membrane will not remain useful as a filter if its porosityis harmfully compromised by the addition of an azlactone-functionalmoiety to its surfaces.

Monomeric 2-alkenyl-1,3-oxazolin-5-ones (which compounds and homologsthereof are referred to herein as 2-alkenyl azlactones) and copolymersthereof are known. Copolymers of 2-alkenyl azlactones and olefinicallyunsaturated monomers and coatings thereof are disclosed in U.S. Pat. No.3,583,950 (Kollinsky et al.). Also, copolymers consisting essentially ofa 2-alkenyl azlactone and an acrylic acid ester, and copolymerizationthereof with vinylidene compounds having at least one hydroxyl group aredisclosed in U.S. Pat. Nos. 3,488,327 and 3,598,790 (both to Kollinskyet al.). U.S. Pat. No. 4,695,608 (Engler et al.) discloses a bulkpolymerization process for free radical polymerization of a vinylmonomer and a monomeric alkenyl azlactone or a macromolecular monomerwith a molecular weight of less than about 30,000 in a wiped surfacereactor such as a twin-screw extruder. Free radical initiator systemscomprising a combination of reagents are useful in the process.Incorporation of alkenyl azlactones into acrylate pressure-sensitiveadhesives improves the adhesives. Also disclosed in this patent aremethods of preparation of 2-alkenyl azlactone monomers.

SUMMARY OF THE INVENTION

This invention provides azlactone-functional surfaces on a pre-existingsupport and methods of preparing such surfaces in a manner which retainsuseful physical and chemical characteristics of the pre-existingsupport. This invention also provides an adduct support prepared fromsuch azlactone-functional support and methods of preparing such adductsupports.

The invention provides a chemically reactive support comprising apre-existing support having surfaces and azlactone-functional moietiescontacting the surfaces and modifying reactivity of such surfaces whileretaining useful physical and chemical characteristics of thepre-existing support.

The invention also provides a method of preparing anazlactone-functional support, comprising the steps of (a) exposingsurfaces of a pre-existing support with high energy radiation togenerate free radical reaction sites on the surfaces and (b) causingazlactone-functional moieties to react with the free radical reactionsites to modify chemical reactivity of the pre-existing support.

The invention also provides a method of preparing anazlactone-functional support, comprising (a) covering surfaces of apre-existing support with azlactone-functional monomers, crosslinkingmonomers, and optionally co-monomers, and (b) copolymerizing themonomers to form a crosslinked, polymerized, azlactone-functionalmoieties at surfaces of the pre-existing support to modify chemicalreactivity of the pre-existing support.

The invention also provides an adduct support, comprising a chemicallyreactive support described above, having azlactone-functionality atsurfaces of the support and a ligand comprising a nucleophilic reagentreacted with the azlactone-functionality.

A feature of the present invention is that methods of preparing theazlactone-functional modified surfaces do not compromise useful physicaland chemical characteristics of the pre-existing support.

Another feature of the present invention is that azlactone-functionalmoieties are present only at surfaces of the pre-existing support,making efficient use of valuable azlactone-functionality.

Azlactone-functional modified surfaces of a pre-existing support areuseful in surface-mediated or catalyzed reactions for synthesis orsite-specific separations. Nonlimiting examples of such uses includeaffinity separation of biomolecules from culture media, diagnosticsupports, and enzyme membrane reactors. Azlactone-functional modifiedsurfaces are capable of covalently binding azlactone-reactive,nucleophilic groups, such as Protein A, which is a biologically activematerial which reversibly binds to an antibody, such as ImmunoglobulinG.

One method of the present invention involves the irradiation of surfacesof a pre-existing support with high-energy radiation to prepare freeradical reaction sites on such surfaces upon which azlactone-functionalmoieties can be formed by homopolymerization, copolymerization, orgrafted reaction with free radically reactive azlactone-functionalmoieties.

Another method of the present invention involves the polymerization orcopolymerization of azlactone-functional moieties as crosslinkedcoatings on surfaces of pre-existing supports.

Another method of the present invention involves the the dispersionpolymerization of azlactone-functional moieties to produce crosslinkedazlactone-functional particles within the pores and interstices of apre-existing porous support.

"Azlactone" means oxazolinone moieties of Formula I: ##STR1## wherein R¹and R² independently can be an alkyl group having 1 to 14 carbon atoms,a cycloalkyl group having 3 to 14 carbon atoms, an aryl group having 5to 12 ring atoms, an arenyl group having 6 to 26 carbon atoms and 0 to 3S, N, and nonperoxidic O heteroatoms, or R¹ and R² taken together withthe carbon to which they are joined can form a carbocyclic ringcontaining 4 to 12 ring atoms, and n is an integer 0 or 1.

"Pre-existing support" means a matrix having surfaces not directlycapable of forming covalent chemical bonds with nucleophilic reagents,especially biologically active materials.

"Surfaces" means both outer surfaces of a support and any applicableinterior surfaces forming pores and interstices within a porous support.

"Biologically active material" means a chemical composition havingnucleophilic-functional groups and capable of reacting in a manner whichaffects biological processes.

"High energy radiation" means radiation of a sufficient dosage andenergy to cause the formation of free radical reaction sites on surfacesof supports. High energy radiation can include electron-beam radiation,gamma radiation, ultraviolet (uv) radiation, plasma radiation, andcorona radiation.

It is an advantage of the present invention that only surfaces of apre-existing support are chemically-modified, such that preciousazlactone-functional moieties are not wasted within the bulk of a matrixof a support being formed in the presence of azlactone-functionalmaterials.

It is another advantage of the present invention that surfaces of apre-existing support are not physically and chemically modified in amanner which diminishes beyond usefulness the physical and chemicalcharacteristics for which the pre-existing support was originallyselected.

Thus, the present invention retains the benefits of the physical andchemical characteristics of the bulk properties of a pre-existingsupport while adding a chemical modification of azlactone-functionalityto surfaces of a support which renders a pre-existing support useful inways an unmodified support could not achieve.

In particular, the presence of azlactone-functionality allows for thecovalent attachment, without intermediate chemical activation of thesupport, of nucleophilic-functional-group-containing materials,especially biologically active materials. Attachment of such materials,without intermediate chemical activation of the support, can provideutility as adsorbants, catalysts, reagents, complexing agents, orpurification supports.

EMBODIMENTS OF THE INVENTION Pre-existing Supports

Selection of a matrix to serve as a support can vary widely within thescope of the invention. A support can be porous or nonporous, dependingon preferred final use. A support can be continuous or non-continuousdepending on ultimate desired usage. A support can be made of a varietyof materials, including supports made of ceramic, glassy, metallic, orpolymeric materials or combinations of materials. A support can beflexible or inflexible depending on ultimate desired usage. Provision ofazlactone-functionality on surfaces of such pre-existing supports doesnot adversely affect the bulk properties of the pre-existing support,other than providing azlactone-functionality which can react withvarious nucleophilic reagents without intermediate chemical activation.

Preferred matrices include polymeric supports, such as woven andnonwoven webs (such as fibrous webs), microporous fibers, andmicroporous membranes.

Webs

Woven and nonwoven webs are useful as supports having either regular orirregular physical configurations of surfaces from whichazlactone-functional moieties can extend. Fibrous webs are particularlydesired because such webs provide large surface areas, with nonwovenfibrous webs being preferred due to ease of manufacture, low materialcost, and allowance for variation in fiber texture and fiber density. Awide variety of fiber diameters, e.g., 0.05 to 50 micrometers, can beused. Web thickness can be varied widely to fit the application, e.g.,0.2 micrometer to 100 cm thick or more.

Fibrous webs can be prepared by methods known in the art, or bymodifications of methods known in the art. Nonwoven webs can be preparedby melt-blowing as is known to those skilled in the art and disclosedin, for example, U.S. Pat. No. 3,971,373, which is incorporated hereinby reference. In general, a molten polymeric material is extruded insuch a way as to produce a stream of melt blown polymer microfibers. Thefibers are collected on a collection screen, with the microfibersforming a web.

The web optionally can be molded or pressed at a pressure of up to 90psi to provide an article having a Gurley number of at least 2 seconds,as described in coassigned U.S. Pat. Nos. 5,328,758 and 5,415,779,incorporated by reference herein.

The nonwoven webs can also optionally include a permeable support fabriclaminated to one or both sides of the web, as described in U.S. Pat. No.4,433,024, or can additionally contain reinforcing fibers as describedin U.S. Pat. Nos. 4,681,801 and 4,868,032, all of which patents areincorporated by reference herein.

The preferred materials useful to prepare nonwoven fibrous webs includepolymers and copolymers of monomers which form fibrous webs. Suitablepolymers include polyalkylenes such as polyethylene and polypropylene,polyvinyl chloride, polyamides such as the various nylons, polystyrenes,polyarylsulfones, poly(vinyl alcohol), polybutylene, poly(ethylene vinylacetate), polyacrylates such as polymethyl methacrylate, polycarbonate,cellulosics such as cellulose acetate butyrate, polyesters such aspoly(ethylene terephthalate), polyimides, and polyurethanes such aspolyether polyurethanes, and combinations thereof.

Nonwoven webs can also be prepared from combinations of co-extrudedpolymers such as polyesters and polyalkylenes. Copolymers of themonomers which provide the above-described polymers are also includedwithin the scope of the present invention.

Nonwoven webs can also be combined webs which are an intimate blend offine fibers and crimped staple fibers.

Fibers and Membranes

Pre-existing, polymeric supports can also include microporous membranes,fibers, hollow fibers, or tubes, all of which are known in the art.

The same materials useful for preparing webs are also suitable forpreparing fibers and membranes. Preferably, membranes are composed ofhomopolymers and copolymers of polyolefins. Nonlimiting examples of suchpolyolefins are polyethylene, polypropylene, polybutylene, andcopolymers of ethylene and vinyl acetate.

A preferred technique useful for preparation of microporousthermoplastic polymeric supports is thermally induced phase separationwhich involves melt blending a thermoplastic polymer with immiscibleliquid at a temperature sufficient to form a homogeneous mixture,forming an article from the solution into a desired shape, cooling theshaped article so as to induce phase separation of the liquid and thepolymer and to ultimately solidify the polymer, and removing at least asubstantial portion of the liquid leaving a microporous polymer matrix.This method and the preferred compositions used in the method aredescribed in detail in U.S. Pat. Nos. 4,957,943; 4,539,256; and4,726,989, which are incorporated herein by reference.

Alternatively, polymeric supports can also be hydrophobic polyolefinmembranes prepared by thermally induced phase separation techniques, butalso having a hydrophilic polymeric shell interlocked about suchhydrophobic membrane surfaces. Coassigned U.S. patent application Ser.No. 08/122,807, the disclosure of which is incorporated by reference,discloses methods of preparation of such hydrophilized, microporousmembranes using poly(vinyl alcohol) precursors to form an extremely thinpoly(vinyl alcohol) shell about the polyolefin surfaces.

Alternatively, polymeric supports can be constructed from poly(vinylalcohol), prepared using poly(vinyl alcohol) precursors, to formhydrogel materials, such as disclosed in U.S. Pat. Nos. 4,528,325 and4,618,649, the disclosures of which are incorporated by referenceherein. Alternatively, polymeric supports can be constructed frompoly(methyl methacrylate) to form other hydrogel materials. Poly(methylmethacrylate) is commercially available and is often used in opthalmicdevices such as intraocular lenses, contact lenses, and the like.

Alternatively, polymeric supports can also be prepared by solvent phaseinversion polymerization techniques. Such techniques are disclosed inU.S. Pat. No. 5,006,247, the disclosure of which is incorporated byreference therein.

Other Supports

Ceramic supports, glass supports, and metallic supports are all known inthe art and are commercially available or can be prepared by a varietyof known techniques.

Azlactone-functional Moieties

Azlactone-functional moieties can be any monomer, prepolymer, oligomer,or polymer containing or comprising azlactone functionality of Formula Iabove and also comprising a site for free radical reaction. Preferably,such reaction site is a vinylic group on an unsaturated hydrocarbon towhich azlactone of Formula I is attached. Such moieties can beindividual azlactone-containing monomers, oligomers formed with freeradical reaction sites and having azlactone-functionality derived fromindividual azlactone-containing monomers, or polymers havingazlactone-functionality, derived from individual azlactone-containingmonomers, and at least one free radical reacting site.

Azlactone-containing Monomers

Preferably, azlactone-functionality is provided by 2-alkenyl azlactonemonomers. The 2-alkenyl azlactone monomers that can be grafted to orpolymerized on surfaces of pre-existing supports are known compounds,their synthesis being described for example in U.S. Pat. Nos. 4,304,705;5,081,197; and 5,091,489 (all Heilmann et al.) the disclosures of whichare incorporated herein by reference.

Suitable 2-alkenyl azlactones include:

2-ethenyl-1,3-oxazolin-5-one,

2-ethenyl-4-methyl-1,3-oxazolin-5-one,

2-isopropenyl-1,3-oxazolin-5-one,

2-isopropenyl-4-methyl-1,3-oxazolin-5-one,

2-ethenyl-4,4-dimethyl-1,3-oxazolin-5-one,

2-isopropenyl-4,4-dimethyl-1,3-oxazolin-5-one,

2-ethenyl-4-methyl-4-ethyl-1,3-oxazolin-5-one,

2-isopropenyl-4-methyl-4-butyl-1,3-oxazolin-5-one,

2-ethenyl-4,4-dibutyl-1,3-oxazolin-5-one,

2-isopropenyl-4-methyl-4-dodecyl-1,3-oxazolin-5-one,

2-isopropenyl-4,4-diphenyl-1,3-oxazolin-5-one,

2-isopropenyl-4,4-pentamethylene-1,3-oxazolin-5-one,

2-isopropenyl-4,4-tetramethylene-1,3-oxazolin-5-one,

2-ethenyl-4,4-diethyl-1,3-oxazolin-5-one,

2-ethenyl-4-methyl-4-nonyl-1,3-oxazolin-5-one,

2-isopropenyl-4-methyl-4-phenyl-1,3-oxazolin-5-one,

2-isopropenyl-4-methyl-4-benzyl-1,3-oxazolin-5-one, and

2-ethenyl-4,4-pentamethylene-1,3-oxazolin-5-one,

The preferred 2-alkenyl azlactones include2-ethenyl-4,4-dimethyl-1,3-oxazolin-5-one (referred to herein as VDM)and 2-isopropenyl-4,4-dimethyl-1,3-oxazolin-5-one (referred to herein asIDM).

If a copolymer is to be formed, a co-monomer having similar or differentchemical or physical properties can be included, depending on thedesired characteristics for the graft or coating. Nonlimiting examplesof co-monomers useful to be copolymerized with azlactone-functionalmoieties to form grafts or coatings include hydroxyethyl methacrylate(HEMA), vinyl acetate, or any of vinyl aromatic monomers; alpha,beta-unsaturated carboxylic acids or their derivatives or vinyl esters;vinyl alkyl ethers; olefins; N-vinyl compounds; vinyl ketones; or vinylaldehydes. Nonlimiting examples of such co-monomers are disclosed inEuropean Patent Publication 0 392 735, the disclosure of which isincorporated by reference.

Preferably, HEMA is used as a co-monomer in order to imparthydrophilicity to the azlactone-functional surface, in order tofacilitate coupling of hydrophilic nucleophilic reagents to form adductsupports.

Such azlactone-functional monomers can be combined for copolymerizingwith non-azlactone-functional monomers in any combination of weightpercentages to control the reaction results.

For example, using a co-monomer of similar reactivity ratio to that ofVDM will result in a random copolymer chain grafted to the free radicalreaction site of the support.

Determination of reactivity ratios for copolymerization are disclosed inOdian, Principles of Polymerization, 2nd Ed., John Wiley & Sons, p.425-430 (1981), the disclosure of which is incorporated by referenceherein.

Alternatively, use of a co-monomer having a higher reactivity to that ofVDM will result in a block copolymer chain grafted to the reaction site,with little or no azlactone-functional moieties near the reactivesurface but considerable azlactone-functionality near the terminus ofthe chain. This construction places azlactone-functionality away fromsurfaces of the support (where steric hindrance might prevent thecoupling of the azlactone-reactive nucleophilic reagent), but covalentlybonded thereto for considerable reactivity with nucleophilic reagents.

Oligomers and Polymers

Although not as preferred as monomers, azlactone-functional prepolymersor oligomers and polymers or copolymers having at least onefree-radically polymerizable site can also be utilized for providingazlactone-functionality on surfaces of a pre-existing support.

Azlactone-functional oligomers and polymers for example, are prepared byfree radical polymerization of azlactone monomers, optionally withco-monomers as described in U.S. Pat. Nos. 4,378,411 and 4,695,608,incorporated by reference herein.

Polymers having azlactone-functional side chains can be prepared byreactive extrusion grafting of azlactone monomers tonon-azlactone-containing polymers, using such techniques as disclosed inEuropean Patent Publication 0 392 783 (Coleman et al.) incorporated byreferences herein.

Nonlimiting examples of azlactone-functional oligomers and prepolymersare disclosed in U.S. Pat. Nos. 4,485,236 and 5,081,197, and EuropeanPatent Publication 0 392 735, all incorporated by reference herein.

In order to be useful in the present invention, theseazlactone-functional polymers and prepolymers must be modified so as toalso comprise at least one free-radically polymerizable site. This isreadily accomplished by reacting a portion of the azlactone-functionalgroups with an ethylenically unsaturated nucleophilic compound, such asthose compounds disclosed in U.S. Pat. No. 4,378,411 identified above,thereby producing a polymer or prepolymer having bothazlactone-functionality and free-radically reactive functionality. Theratio of azlactone moieties to unsaturated moieties can vary from 99:1to 1:99, although it is preferable for the azlactone moiety content inthe polymer or prepolymer to be at least fifty percent (50%).

Method of Providing Azlactone-Functional Surfaces on Pre-existingSupports

In general, processes for providing the azlactone-functional supports ofthe present invention involve exposing a pre-existing support,especially a pre-existing, polymeric support, to high energy radiationand to free-radically polymerizable azlactone-functional moieties.Exposure of a support to an azlactone-functional moiety can take placeeither simultaneously with or subsequent to the irradiation of thesupport. Depending on the the type of radiation and other processconditions, the azlactone-functional polymer which is produced can beeither grafted to the surface of the pre-existing support or can beformed as a coating on the support or can become particles enmeshedwithin void spaces of the support. In the former instance, theazlactone-functional moiety becomes covalently linked to the support,whereas in the latter two instances, it does not. Regardless, thepre-existing support is transformed into being capable of formingchemical bonds with nucleophilic reagents, especially biologicallyactive materials.

Methods of Irradiation

Pre-existing supports are subjected to radiation from a high-energysource to form free radical sites on or near surfaces of such supports.In the case of nonpolymeric supports, no free radical sites are formedon surfaces. However, during plasma or corona treatment, free radicalsites are formed from the monomer molecules adsorbed onto thenonpolymeric support surfaces. High energy radiation can be classifiedfor the purposes of the present invention as either penetrating ornon-penetrating. Penetrating radiation is utilized when one wants toprovide azlactone-functionality to both the interior and exteriorsurfaces of a pre-existing support, whereas non-penetrating radiation isuseful to provide azlactone-functionality only to the outer surfaces ofthe pre-existing support.

Nonlimiting examples of penetrating radiation include beta, gamma,electron-beam, x-ray, uv and other electromagnetic radiation, whereasnon-penetrating radiation includes alpha, plasma, and corona radiation.In some instances, corona radiation can be become penetratingirradiation.

Penetrating Irradiation

Many forms of penetrating radiation are of sufficiently high energy,that when absorbed by a pre-existing support, sufficient energy istransferred to that support to result in the cleavage of chemical bondsin that support. Homolytic chemical bond cleavage results in theformation of a free radical site on the support. Thus, this type ofradiation is useful when it is desired to covalently link theazlactone-functional moieties, via a free radical grafting reaction, tothe surfaces of a pre-existing support. Electron beam and gammaradiation are preferred for this method of grafting due to theready-availability of commercial sources.

It should be noted that, although penetrating radiation also generatesfree radical sites within the bulk of many supports, these sites aregenerally not as available for reactions with azlactone-containingmoieties because such moieties are less likely to diffuse into the bulkof a support than react at an outer surface of that support. Thus, evenwith penetrating radiation to generate reaction sites,azlactone-functionality is usually found principally at outer surfacesof a support.

Sources of electron-beam radiation are commercially available, includingan Energy Sciences Inc. Model CB-150 Electrocurtain Electron BeamProcessor. Sources of uv radiation are high and medium pressure mercurylamps, deuterium lamps, and "blacklights" emitting 180 nm to 400 nm(with preferred maximum intensity at about 360 nm) light, which arecommercially available from a number of vendors, including GeneralElectric Company and GTE Sylvania. Sources of gamma irradiation arecommercially available from Atomic Energy of Canada, Inc. using acobalt-60 high-energy source.

High energy radiation dosages are measured in megarads (Mrad) orkilograys (kGy), which is 1/10 of a mRad. Doses can be administered in asingle dose of the desired level or in multiple doses which accumulateto the desired level. Dosages can range cumulatively from about 10 kGysto about 200 kGys and preferably from about 30 kGys to about 100 kGys.Preferably, the cumulative dosage exceeds 30 kGys (3 Mrads).

Supports can be irradiated in a package or container where thetemperature, atmosphere, and other reaction parameters can becontrolled.

Temperature can be ambient temperature.

The atmosphere can be air or preferably an inert atmosphere such asnitrogen.

The pressure in the container can be atmospheric, elevated or depressedto a partial or complete vacuum. Preferably it is atmospheric.

Depending upon the control of the irradiation conditions, supports canbe irradiated in a batch or continuous process.

After irradiation and prior to contact with the azlactone-functionalmoiety, the atmosphere around the surfaces should be kept free offree-radically reactive substances, especially O₂.

After the first step where irradiation forms free radical reactionsites, the second step provides azlactone-functional moieties to reactwith such sites under suitable free radical reaction conditions.

Generally, irradiation can take place in the presence or absence of theazlactone-functional moieties. When conducted in the presence ofazlactone-functional moieties, ungrafted free-radical (co)polymerizationcan occur in addition to grafting polymerization. As a consequence, itcan be preferred to irradiate a pre-existing support in the absence ofazlactone-functional moieties followed by contacting the irradiatedsupport with azlactone-functional moieties to initiate the desired freeradical grafting reaction. This may be accomplished by immersing thesupport in, coating the support with, or spraying the support withvapors, dispersions, or solutions containing azlactone-functionalmoieties. Alternatively, production of water-solubleazlactone-functional polymers can be minimized during irradiation in thepresence of azlactone-functional moieties by incorporation of amultifunctional cross-linking monomer.

Another method of radiation-induced grafting involves irradiation of apolymer film with ionizing radiation in the presence of ambient oxygento generate hydroperoxide functionality on the surface. The peroxidesare then used to initiate graft-polymerization of olefinic monomers bythermally induced free radical polymerization, according to techniquesdisclosed in Gupta et al., Eur. Polym. J., 25 (11), 1137 et seq. (1989).Alternatively, hydroperoxide species can be used to initiate graftpolymerization, according to techniques disclosed in Yamauchi et al., J.Appl. Polym. Sci., 43, 1197 et seq. (1991).

Ultraviolet radiation, which is a penetrating radiation for purposes ofthe present invention, is different from other penetrating radiations inthat uv radiation does not provide enough energy directly to mostsupports to produce free radical sites. Therefore, uv radiation isgenerally conducted in the presence of both azlactone-functionalmoieties and photoinitiators, which absorb light in the uv-visible range(250-450 nm) and convert this light energy to chemical energy in theform of free radical species. Generation of free radicals byphotoinitiators generally occurs by one of two processes, intramolecularbond cleavage or intermolecular hydrogen abstraction. Suitablephotoinitiators are identified in Oster et al., "Photopolymerization ofVinyl Monomers" Chem Rev., 68, 125 (1968), the disclosure of which isincorporated by reference. Nonlimiting examples include acyloins andderivatives thereof; diketones; organic sulfides; S-acyldithiocarbamates; phenones; sulfonyl halides; and azo compounds. Ofthese possible photoinitiators, azobis(isobutyronitrile), acyloins,acyloin ethers, and benzil ketals and1-phenyl-2-hydroxy-2-methyl-1-propanone (commercially available asDarocure™ 1173 brand photoinitiator from E Merck) are preferred.

The manner in which the azlactone-functional surface is imparted to thepre-existing support can be influenced by the choice of photoinitiator.Whereas most photoinitiators will promote free radical(co)polymerization of azlactone-functional moieties to produce coatings,those initiators which are prone to abstraction reactions, particularlyphenones, result in simultaneous grafting to the pre-existing support.It is preferred to utilize crosslinking comonomers with uv irradiationto minimize the production of soluble polymer.

A support can be immersed in, sprayed with, dipped into, or otherwisecontacted with a mixture, dispersion or solution of azlactone-containingmonomers, photoinitiator, and optionally a crosslinking monomer and/ornon-azlactone-containing co-monomer(s). Then, the coated support isexposed to uv radiation to cure the monomers, thus resulting in theformation of azlactone-functional copolymer as a continuous ordiscontinuous coating on surfaces of the support.

After rinsing to remove unreacted monomers and drying, anazlactone-functional support is available for nucleophilic reaction.

Nonlimiting examples of crosslinking monomers for theseazlactone-functional coatings include ethylene glycol dimethylacrylate(EGDMA), trimethylolpropane trimethacrylate (TMPTMA),methylenebisacrylamide (MBA), and divinylbenzene.

Nonlimiting examples of co-monomers include hydroxyethyl methacrylate(HEMA), butyl acrylate (BA), isooctyl acrylate (IOA), butyl methacrylate(BMA), and isobutyl methacrylate (IBMA).

In some instances, the azlactone-functional copolymer is deposited assmall particles or aggregates of small particles contacting the surfacesor otherwise within the porous structure of the pre-existing support.

Coating and uv photopolymerization can occur in ambient conditions.

Temperature can be about -78° C.-100° C. and preferably is ambient.

Atmospheric conditions need to be inert using non-oxygen gases andpreferably is nitrogen or a noble gas such as argon. Alternatively, aweb coated with the desired monomer solution can be placed between twooxygen-occluding sheets that are transparent to the desired type ofradiation.

Since free radical reactions occur quickly, the contact time of theirradiated support with the azlactone-functional moiety ranges frommomentary to less than 30 min., depending on radiation intensity.Reaction times as short as a few seconds are often enough to providecompleted reaction.

Non-Penetrating Irradiation

Plasma and corona radiation differ from penetrating irradiationtechniques because only the outer surfaces of a pre-existing support aresubjected to treatment with vaporous, excited azlactone-functionalmoieties. This method of irradiation grafting only requires one step.

Electrical energy in the form of plasma discharge (also known as glowdischarge) or corona discharge activates the azlactone-functionalmoieties in the vapor state for contact with the outermost surfaces ofthe support. The outermost surfaces can include adsorbed monomermolecules. Without being bound to a particular theory, it is believedthat the excited azlactone-functional moieties react with surface freeradical sites leading to the deposition of a thin film or networkcoating the support.

Even though ethylenically unsaturated monomers are not required fornon-penetrating radiation methods, preferably, suitableazlactone-functional moieties are monomeric and covalently react withfree radical sites on the supports.

As with penetrating irradiation techniques, one can control the natureof the azlactone-functionality formed by employing various amounts ofazlactone-functional moieties and non-azlactone-functional moieties andby introducing such amounts into the reaction vessel at different times.

For example, one can form a corona-treated support having a crosslinkedcoating or network of VDM and HEMA covalently bound thereto.Alternatively, one can form a plasma-treated support having layers ofdeposited HEMA and VDM extending from the support. Alternatively, onecan treat regiospecific surfaces of a pre-existing support. Bypreventing certain portions of surfaces from being subjected to coronaor plasma discharge treatment, one can produce supports having specificregions of azlactone-functionality.

Alternatively, one can treat regiospecific surfaces sequentially withdifferent azlactone-functional moieties to produce a complex surface ofa support for multiple or differentiating nucleophilic reactions.

Sources of plasma discharge energy operate typically at DC, AC, high,radio, or microwave frequencies. Such sources are commercially availablefrom a number of vendors including ENI Power Systems, Inc. Theexcitation frequency is typically 0-2.5 GHz, preferably 25-125 kHz. Thepower density at the support's surface is typically 1×10⁻³ -0.4 W/cm²where the normalization is based on the projected area of the support(as opposed to its actual surface area, if porous). Preferably, thepower density is 0.01-0.05 W/cm². The gas/vapor composition comprisesazlactone-functional moieties, either pure or mixed with other organicor inorganic vapors or gases. Nonlimiting examples of such vapors orgases include He, Ar, NO₂, CO, and CO₂ ; alkanes, alkenes, alkynes;functionalized alkanes, alkenes, and alkynes; acrylates, methacrylates;and other comonomer candidates identified above with respect tocopolymerization of azlactone-functional moieties.

Sources of corona discharge energy are available commercially from anumber of vendors, including ENI Power Systems, Inc. The excitationfrequency is typically 5-100 kHz, preferably 10-50 kHz.

The pressure is typically 0.5-5 atmospheres, preferably at or near 1atmosphere.

The power density is typically 0.5-6 W/cm², preferably 1-3 W/cm², whenapplying the same normalization of surface area as described withrespect to plasma discharge above.

The amount of deposition of azlactone-functional moieties can becontrolled by the amount of time exposed to discharge. The amount oftime using the above power densities can range from 0.05 secs to severalhours, and preferably from about 1 second to about 5 minutes.

The gas/vapor composition comprises azlactone-functional moieties mixedwith other organic or inorganic gases or vapors, from among thecandidates described above with respect to plasma discharge. Preferably,the azlactone-functional moieties have a partial pressure of 1-100mTorr.

It has recently been published in European Patent Publication 0 467 639(1991) that a process believed to involve corona discharge caneffectively achieve penetrating irradiation effect on nonwoven materialusing a Helium atmosphere and dielectric protection over both electrodesof the corona discharge apparatus. With this technique, one can employcorona discharge of azlactone-functional moieties to render interiorsurfaces of a porous support azlactone-functional. Power densities andtime of discharge described above for non-penetrating irradiation neednot change.

Adduct Supports and Usefulness of the Invention

Because azlactone-functional moieties occupying a surface of apre-existing support are capable of multiple chemical reactions,azlactone-functional modified surfaces of the present invention can formadduct supports.

Once covalently bonded to or otherwise coating a surface, electrophilicazlactone-functional moieties can react through a nucleophilic ringopening reaction at the carbonyl group with any of a myriad ofnucleophilic reagents. The result is the formation of an adduct supporthaving specific reactivities determined by the nature of thenucleophilic reagent employed in the reaction.

Nonlimiting examples of nucleophilic reagents include biologicallyactive materials, acids, bases, chelators, hydrophiles, lipophiles,hydrophobes, zwitterions, detergents, and any other chemical which canreact with the azlactone-functionality to confer on the surfaces of thepre-existing support a modified reactivity which differs from that whichexisted on the support prior to azlactone-functionality modification.For example, one can modify a hydrophobic surface by reacting onazlactone-functional adduct support with a nucleophilic, hydrophilicmoiety. Examples of nucleophilic, hydrophilic compounds includepoly(ethylene oxide) commercially available as Jeffamines from Texaco,Inc.

Thus, surfaces of a support can become azlactone-functional and thenadduct-reactive, without loss of the physical and chemicalcharacteristics of such supports such as porosity, flux, color, surfacearea, permeability, solvent resistance, hydrophilicity, flexibility,mechanical integrity, and other stability or feature in the useenvironment. Unexpectedly, pre-existing supports can add all of thebenefits of azlactone-functionality without an effective diminution ofthe physical and chemical characteristics of bulk properties of thepre-existing support.

Ligands and Adduct Supports

Adduct supports have ligands coupled or otherwise tightly bound toazlactone-functional moieties extending from surfaces of supports toform biologically or chemically active reaction sites. For directcoupling, nonlimiting examples of nucleophilic ligands include primaryand secondary amines, alcohols, and mercaptans. Of these,amine-functional ligands are especially preferred.

While not being limited to a particular theory, it is believed that aligand forms a covalent bond when coupled to an azlactone-functionalmoiety.

Ligands useful for the preparation of adduct supports can also varywidely within the scope of the present invention. Preferably, a ligandis chosen based upon the contemplated end use of the adduct support.

Once ligands are coupled to azlactone-functional grafts or coatings,such ligands are available for biological or chemical interaction, suchas adsorbing, complexing, catalysis, or reagent end use.

Adduct supports are useful as adsorbants, complexing agents, catalysts,reagents, as enzyme and other protein-bearing supports, and aschromatographic articles.

In a preferred aspect of the present invention, the ligand desired forcoupling is a biologically active material having azlactone-reactive,nucleophilic-functional groups. Nonlimiting examples of biologicallyactive materials are substances which are biologically,immunochemically, physiologically, or pharmaceutically active. Examplesof biologically active materials include proteins, peptides,polypeptides, antibodies, antigenic substances, enzymes, cofactors,inhibitors, lectins, hormones, receptors, coagulation factors, aminoacids, histones, vitamins, drugs, cell surface markers, and substanceswhich interact with them.

Of the biologically active materials, proteins, enzymes and antigenicsubstances are desired for coupling to azlactone-functional supports.Nonlimiting examples of proteins, enzymes, and antigenic substancesinclude natural and recombinant Protein A (ProtA), Immunoglobulins suchas rat (rIgG), human (hIgG), bovine (bIgG), rabbit (rbIgG), and mouse(mIgG), Concanavalin A (ConA), Bovine Serum Albumin (BSA), Thyroglobulin(TG), Apoferritin (Af), Lysozyme (Ly), Carbonic Anhydrase (CA), Lipase,Pig Liver Esterase, Penicillin acylase, and Bacterial Antigen (BA). Usesfor immobilized proteins, enzymes and antigenic substances are disclosedin European Patent Publication 0 392 735.

A presently preferred biologically active material is ProtA because ofits multitude of uses in bioseparations.

Alternatively, an adduct support of the present invention can comprise acoupled enzyme to catalyze a chemical transformation of substancesrecognized by the enzyme. Also, a support comprising a coupled antigenicsubstance can be utilized for affinity purification of a correspondingantibody from a complex biological fluid flowing through the porousmatrix of the adduct support. In other examples, an adduct supporthaving Protein A coupled to internal and external surfaces can adsorbbiologically active materials such as Immunoglobulin G for affinityseparations processes. In other examples, an adduct support can be usedfor immobilization of antibodies or be used for immunodiagnostics or forWestern blotting.

Alternatively, the ligand can be a hydrophile for improvingcompatibility of mammalian body implants, such as intraocular lenses,with adjoining tissues. One example of a ligand especially suitable forchemically modifying body implants is an anticoagulant, such as achemically-modified heparin, e.g., an amine-terminated heparin.

Azlactone-functional moieties will undergo nucleophilic attack byamines, thiols, and alcohols. Thus, ligands having at least one amine,thiol, or alcohol group thereon are candidates for coupling toazlactone-functional surfaces. Amine-functional ligands are preferreddue to ease of reaction and stability of the linkage so formed.

Coupling of ligands to preferred azlactone-functional surfaces can usemethods of using inorganic or organic polyanionic salts in suchconcentrations as to achieve high broad specific biological activity forthe coupled ligand, such as methods disclosed in coassigned U.S. Pat.No. 5,200,471, the disclosure of which is incorporated by reference.

Coupling of ligands to preferred azlactone-functional surfaces accordingto the present invention results in adduct supports having the formula##STR2## wherein R¹ R² and n are as previously defined, R³ is H or CH₃,

X can be --O--, --S--, --NH--, or --NR⁴ wherein R⁴ can be alkyl or aryl,and

G is the residue of HXG which performs the adsorbing, complexing,catalyzing, separating, or reagent function of the adduct support.

HXG is a nucleophilic reagent and can be a biologically active material,dye, catalyst, reagent, and the like.

Ligands having azlactone-reactive, amine, hydroxy, or thiol nucleophilicfunctional groups react, either in the presence or absence of suitablecatalysts, with azlactone-functional groups by nucleophilic addition asdepicted in the equation. ##STR3## wherein R¹, R², R³, n, X, and G areas previously defined.

Depending on the functional group present in the ligand, catalysts maybe required to achieve effective attaching reaction rates. Primary aminefunctional groups require no catalysts. Acid catalysts such astrifluoroacetic acid, ethanesulfonic acid, toluenesulfonic acid, and thelike are effective with hydroxy and secondary amine functional groups.

In other aspects of the invention, the ligand is not biologically activebut has other properties which lead to its end use. For example, theligand can contain ionic functional groups. In that event, the resultantadduct article may be utilized in ion exchange type applications.Suitable ionic groups include carboxylic acid, sulfonic acid, phosphonicacid, tertiary amine, and quaternary amine groups. Examples of usefulionic group containing ligands include aminocarboxylic, sulfonic, orphosphonic acids such as glycine, alanine, leucine, valine, β-alanine,γ-aminobutyric acid, 1- and 3-aminopropyl-phosphonic acid, taurine,γ-amino octanoic acid, aminomethylphosphonic acid, amino-methanesulfonicacid, and the like; hydroxy-acids such as isethionic acid,3-hydroxy-propane sulfonic acid, lactic acid, glycolic acid,hydroxymethylphosphonic acid, p-hydroxybenzoic acid, and the like; andamino- and hydroxy-functional tertiary and quarternary amines such as2-diethylaminoethylamine, 3-dimethyl-aminopropylamine,N,N-diethylethanol-amine, and the like, and quaternized versionsthereof. When the amine-, hydroxy- or thiol-functional ligand is asimple aliphatic and/or aromatic hydrocarbon, the resultant adductarticle may be useful in reverse phase or hydrophobic interaction typechromatographic processes. Reaction of the support of this inventionwith very hydrophilic or hydrophobic ligands can be used to produceadduct articles displaying highly absorbant properties towards aqueousor oily fluids, respectively. Other types of ligands and uses will beobvious to one skilled in the art and are considered to be within thescope of the present invention.

Objects and advantages of this invention are further illustrated by thefollowing examples, but the particular materials and amounts thereofrecited in these examples, as well as other conditions and details,should not be construed to unduly limit this invention.

EXAMPLES Example 1 Preirradiation Electron Beam Grafting of HydrophobicPolyethylene (PE) Microporous Membrane with 2-Vinyl4,4-dimethylazlactone (VDM)

A PE microporous membrane, prepared according to the method of Example23 of U.S. Pat. No. 4,539,256 (Shipman), incorporated by referenceherein, having a pore size of 0.496 μm, a thickness of 73.9 μm and avoid volume of 81.5%, was passed through an electron beam (e-beam)chamber within a Model 1 Electrocurtain CB-300/30/380 (manufactured byEnergy Sciences, Inc., Wilmington, Mass.) to generate free radicals onthe membrane. The accelerating voltage of the e-beam was 150 KV, withtotal irradiation dose of 50 kGys (5 Mrads). Membrane samples (7.6×12.7cm) were passed through the e-beam equipment taped to a polyestercarrier web traveling at 6.1 m/min.

The samples exited the e-beam chamber directly into a N₂ purged box,where they were removed from the carrier and immersed into a solution ofVDM (SNPE, Princeton, N.J.) dissolved in ethyl acetate. The inertatmosphere in the glove box was intended to prevent premature quenchingof the generated radicals by oxygen. The monomer solutions hadconcentrations of 25, 50, and 100 volume-percent VDM and had been purgedwith argon for 1 h to displace any dissolved oxygen. Irradiatedmembranes were soaked in the monomer solution for 3 to 5 min. followedby a 5 min. soak in pure ethyl acetate to wash out excess monomer. Theywere dried and placed in zip-lock bags to prevent possible hydrolysis ofthe azlactone by atmospheric moisture.

Fourier-transform infrared spectroscopy (FT-IR) was used to characterizethe grafted membranes. The ratio of the azlactone carbonyl absorption(1824 cm-1) to the PE C-H band (1462 cm-1) gives a relative measure ofthe bound azlactone. Ratios of 0.023, 1.78, and 1.27 were found for thesamples reacted with 25, 50, and 100% VDM, respectively.

To confirm that all of the VDM was indeed covalently grafted to themembrane, the samples were extracted by three 15 min. soakings in pureethyl acetate (three replicates of each). Weight loss values were 1.1,0.7, and 0.0%. Since there was a weight gain of at least 10% during thegrafting step, it was concluded that 90% or more of the VDM wascovalently bonded.

COMPARISON EXAMPLE 2 Mutual Irradiation E-Beam Grafting onto PEMicroporous Membranes

As a demonstration of the advantage of preirradiation grafting over thatof mutual irradiation, the same microporous PE membrane as used inExample 1 was saturated with 10% (w/v) VDM/ethyl acetate solution justprior to being passed through the e-beam chamber for exposure at a doseof about 50 kGy. The membrane was rinsed in pure ethyl acetate for about5 min. upon emergence from the e-beam chamber to remove unpolymerizedmonomer. The azlactone:PE ratio by FT-IR was 1.39, indicating that asubstantial amount of the VDM was indeed bound to the membrane. Themembrane weight decreased by 10% following the extended solvent soakingprocedure described in Example 1, indicating that a significant portionof the VDM was not grafted to the membrane. It is also likely thathomopolymers of VDM (not bound to the membrane) accounted for much ofthe VDM which was not readily washed from the membrane because ofentrapment within the membrane.

EXAMPLE 3 Preirradiation E-beam Grafting of Hydrophilized PE MicroporousMembranes

The starting PE microporous membrane from Example 1 was hydrophilized bycoating the internal and external pore surfaces with a 4% (w/v) solutionof poly(vinyl trifluoroacetate) (PVTFA) followed by reaction withammonia gas to convert the PVTFA to a highly crystalline poly(vinylalcohol), a hydrophilic polymer, using the procedures described inExample 7 of coassigned, copending U.S. patent application Ser. No.08/122,709 (Gagnon et al.) published as PCT Publication WO 92/07899).Grafting conditions were the same as described in Example 1 except forthe addition of 30 kGy and 100 kGy treatments. The FT-IR results aregiven in Table 1.

                  TABLE 1                                                         ______________________________________                                        The Effect of Varying the Irradiation Dose and                                Azlactone Concentration on the Ratio of the                                   Azlactone-to-PE IR Signals                                                    FT-IR Ratio (1824:1462 cm-1) at                                               Various Irradiation Doses (kGys)                                                      Dose (kGys)                                                           VDM %     30           50      100                                            ______________________________________                                        25        --           0.38    --                                             50        1.70         1.32    5.06                                           100       1.10         5.23     0.96*                                         ______________________________________                                    

The marked (*) membrane was completely filled with polymer and swelledupon solvent rinse. Upon drying it was too thick for accurate IRmeasurement; thus the ratio is not indicative of the amount of graftedVDM.

A surface area measurement was performed according to the followingmethod: A sample measuring approximately 3 cm×5 cm was placed in a taredsample holder of a Quantasorb BET Surface Area Analyzer (QuantachromeCorp.). The sample was degassed by flushing with helium at 50° C. for 1hr. The sample holder was then immersed in liquid nitrogen, and ahelium/Krypton gas mixture was passed through the sample. At thistemperature, only the Krypton will adsorb onto the surfaces of thesample, thus depleting the Krypton in the gas mixture passed through thesample. The surface area calculation is based upon the assumption thatthe probe gas adsorbs on all available sample surface area in amonolayer; thus the amount adsorbed times the adsorbate cross-sectionalarea is proportional to the specific surface area. The depletion ofKrypton from the mixture, (i.e., the amount of Krypton adsorbed) isdetected with a sensitive thermal conductivity detector. Upon rewarmingof the sample to room temperature, the adsorbed Krypton is released andalso quantified. The amount of adsorbed Kryton and the mass of thesample are used to calculate the spectific surface area/unit mass value.

The above BET surface area measurement was performed on the unirradiatedhydrophilic membrane control and on the 50 kGy-100% sample showed thatthe control value (18.6 m² /g) had been reduced by over 50% to 8.1 m² /gby the grafting of poly(vinylazlactone), which is not deemed to bediminished beyond usefulness of the membrane.

In all instances but one (30 kGy/50%), there was no detectable weightloss following the extended solvent extraction described in Example 1.Of the possibilities, the process employing 50 kGy/25% seemed to be thebest compromise to avoid pore blockage while providing azlactonefunctionality.

EXAMPLE 4 Reaction of Azlactone-Grafted Hydrophilic PE Membrane withAmmonia

Portions of the treated membranes described in Examples 1 and 3, eachprepared using 50 kGy and 50% VDM, were placed in an ammonia atmospherein an enclosed glass vessel by suspending them above a concentrated NH₄OH solution for 10 min. at ambient temperature. FT-IR (using a ModelFTS-40 spectrophotometer, Bio-Rad, Digilab Div., Cambridge, Mass.)measurements of both ammonia-reacted membranes and unreacted controlmembrane showed that the 1824 cm-1 azlactone absorbance band on theammonia-reacted membranes had nearly entirely disappeared and that a newband appeared at 1659 cm-1 which is indicative of an amide bond. Thisconfirmed that virtually all of the azlactone is available for reaction.These results show that almost any type of surface chemistry might beprepared from an azlactone-grafted membrane surface by choosing as asecondary reagent one which has both the desired functionality and anamine functionality.

EXAMPLE 5 E-beam-Grafting of Azlactone to Porous Polyethylene FilmsIncreases the Amount of Coupled Protein

Azlactone-functional and ungrafted control membranes were prepared asdescribed in Example 3. Protein solutions were radiolabeled usingIodo-Beads™ beads (commercially available from Pierce Chem., Rockford,Ill.) and NaI-125 (Dupont NEN, Billerica, Mass.) using the proceduresdescribed in the product insert. Specific radioactivities obtained were:Protein A, (Genzyme, Boston) 2782 cpm/μg; immunoglobulin G (IgG, SigmaChem., St. Louis), 2000 cpm/μg; and bovine serum albumin (BSA, Sigma),2885 cpm/μg.

Circular portions (7 mm diameter) of the membrane were cut out using apaper punch. The membrane discs were then incubated with radiolabeledprotein in 250 μl of 25 mM sodium phosphate, 150 mM NaCl, pH 7.5, for 60min. at ambient temperature. Some membranes were reacted with 3.0Methanolamine, pH 9.0, for 30 min. prior to the protein incubation to"deactivate" the azlactone functionality. Following the proteinincubations all membranes were reacted an additional 15 min. with 500 μlof the ethanolamine reagent to inactivate remaining azlactones as wellas rinse out unbound protein. Each membrane was subsequently rinsed anadditional three times with 500 μl of the phosphate buffer. After thebound radioactivity was determined using a Model 5230 Auto-Gammascintillation counter (Packard, Downers Grove, Ill.), the membranes wereincubated for 4 h at 37° C. in 500 μl of 1.0% sodium dodecylsulfate(SDS) solution followed by determination of residual radioactivity. SDSis a strongly denaturing detergent capable of removing all but the mosttenaciously bound protein. In these experiments, control membranes werecompletely untreated. These and all experiments described in thisexample were performed in triplicate.

                  TABLE 2                                                         ______________________________________                                        The Binding of Three Proteins to E-beam-                                      Grafted and Control Porous Membranes                                                       Total                                                                         Bound                 Coupled                                                 Protein  SDS          Protein                                    Membrane     (μg/cm.sup.2)                                                                       Resistance(%)                                                                              (μg/cm.sup.2)                           ______________________________________                                        With Protein A                                                                Untreated    4.0      14           0.55                                       Untreated Quenched                                                                         4.2      11           0.50                                       Grafted      3.2      58           1.86                                       Grafted Quenched                                                                           2.7      21           0.57                                       With Immunoglobulin G                                                         Untreated    7.2      18           1.24                                       Untreated Quenched                                                                         8.9      11           0.95                                       Grafted      6.1      45           2.74                                       Grafted Quenched                                                                           4.8      27           1.31                                       With Bovine Serum Albumin                                                     Untreated    3.4      18           0.59                                       Untreated Quenched                                                                         3.6      15           0.53                                       Grafted      1.7      68           1.18                                       Grafted Quenched                                                                           1.4      31           0.42                                       ______________________________________                                    

Consistently for all three proteins there was at least a two-foldincrease in the amount of coupled protein as a result of the e-beam/VDMtreatment. This was especially surprising because there is a decrease intotal binding. The cause of the decrease in total binding was uncertainsince this series included no e-beam only or e-beam plus solventcontrols; however, it was presumed that the 50% reduction in totalsurface area (compare with Example 3) is the reason.

Treatment of the membrane with ethanolamine to inactivate the azlactonefunctionality reduced the amount of coupled protein to about the levelof untreated membranes.

EXAMPLE 6 The Binding of Radiolabeled Protein to E-Beam Grafted PorousPolyethylene Membrane

Unless indicated otherwise, all grafting procedures are identical to the50 kGy example described in Example 3. Membranes were treated asdescribed in Example 5. Protein A specific radioactivity was 1884cpm/μg. Membranes were incubated overnight (16 h) with continuousrocking and treatment with ethanolamine was increased to 60 min.Controls were e-beam-treated only and e-beam-treated plussolvent-treated.

                  TABLE 3                                                         ______________________________________                                        Binding Protein A to E-beam Grafted and                                       Control Porous Membranes                                                                     Total                                                                         Bound                Coupled                                                  Protein  SDS         Protein                                   Membrane       (μg/cm.sup.2)                                                                       Resistance(%)                                                                             (μg/cm.sup.2)                          ______________________________________                                        E-beam         9.1      33          3.0                                       E-beam + Solvent                                                                             7.9      25          2.0                                       E-beam + 50% VDM                                                                             7.0      56          3.9                                       E-beam + 100% VDM                                                                            7.4      85          6.4                                       ______________________________________                                    

The solvent treatment caused a 13% decrease in the amount of proteinwhich bound to an e-beam-treated membrane, because of the solventsolubilizing some of the hydrophilizing shell of poly(vinyl alcohol)which is required for the membrane to wet. There was no significanteffect of azlactone on the total binding (which is probably proportionalto the total available surface area); however, there was a pronouncedeffect of azlactone on the quality of the protein binding, i.e., a largeincrease in the amount of the protein which is so tightly bound,i.e.,coupled, that it resists removal by SDS. Although it is not preciselyaccurate to ascribe covalency to the SDS resistant fraction, it ishighly probable that increases in SDS resistance reflect increases incovalent binding.

Also, increasing the incubation time for the protein binding step from 1h (Example 5) to 16 h increased the amount of the total binding 2.5-foldand the amount of coupling 3.5-fold. Thus, a 1 h incubation did notallow for full, but passive diffusion of the protein through themembrane.

EXAMPLE 7 The Effect of VDM Concentration on Protein Binding

Membranes were irradiated with 50 kGy and treated as described inExample 3 and protein coating procedures were identical to those inExample 6. Protein A specific radioactivity was 1767 cpm/μg.

                  TABLE 4                                                         ______________________________________                                        Effect of VDM Concentration on Protein Binding                                               Total                                                                         Bound    SDS     Coupled                                                      Protein  Resist. Protein                                                                              IR                                     Membrane       (μg/cm.sup.2)                                                                       (%)     (μg/cm.sup.2)                                                                     Ratio*                                 ______________________________________                                        E-beam         7.53       25%   1.84   0.0                                    E-beam + solvent                                                                             6.97     21      1.47   0.0                                    E-beam + 25% VDM                                                                             6.55     30      1.97   0.168                                  E-beam + 50% VDM                                                                             6.19     36      2.20   0.638                                  E-beam + 100% VDM                                                                            2.58     67      1.73   0.540                                  ______________________________________                                         *Ratio of the absorbance of 1824 cm1 azlactone band to 1462 cm1               polyethylene band.                                                       

There were two effects of increasing VDM concentrations: 1) Aconcentration-dependent increase in the percent of SDS resistantprotein; 2) a concentration-dependent decrease in the total proteinbinding. These effects combine to yield an optimum amount of coupledProtein A at 50% VDM. These opposing effects were consistent withreduced access of protein caused by exceedingly long chains of poly(VDM)at high VDM concentrations, blocking protein access to the innermembrane surfaces.

EXAMPLE 8 Retention of Biological Activity of Membrane-Bound Protein

It was found that Protein A bound covalently to the porous PE membranethrough azlactone retained its ability to bind human IgG. This wasaccomplished by a two-part experiment: determination of Protein Abinding using radiolabeled Protein A, and, in parallel, determination ofthe amount of radiolabeled IgG bound to membrane-bound unlabeled ProteinA.

Membranes were prepared as described in Example 3. All bindingprocedures were identical to Example 5 except that incubations withProtein A (whether radiolabeled or not) were for 5.5 h. Radioactivitydeterminations were made on those membranes which had been incubatedwith radiolabeled Protein A (1590 cpm/μl specific radioactivity). Thosemembranes which had been bound with unlabeled Protein A were incubatedan additional 16 h with radiolabeled IgG (specific radioactivity: 1500cpm/μg). They were rinsed, and IgG binding was determined by isotopicdecay followed by the SDS step and a repeat of the bindingdetermination. The results from this series of experiments are found inTable 5. The control membranes were e-beam- and solvent-treated.

                  TABLE 5                                                         ______________________________________                                        The Binding of IgG to Protein A-Coupled                                       Membrane                                                                                              Immuno-                                                           Protein A   globulin G                                                          Total             Total                                                       Protein A SDS     IgG    SDS                                                  Bound     Resist. Bound  Resist.                                Membrane      (μg/cm.sup.2)                                                                        (%)     (μg/cm.sup.2)                                                                     (%)                                    ______________________________________                                        Control       7.66      20      12.1   2.9                                    Control + 25% VDM                                                                           6.84      31      10.6   3.4                                    Control + 50% VDM                                                                           5.63      42      9.9    3.5                                    Control + 100% VDM                                                                          3.03      57      3.8    4.4                                    ______________________________________                                    

The higher SDS resistances for the coupling of Protein A to the graftedmembrane (compared to the binding of IgG to the Protein A-derivatized,grafted membrane) showed that Protein A was bound covalently to themembrane and IgG was bound non-covalently to the Protein A. The SDSresistances of 3-4% indicate that the VDM was almost completelyinactivated. As observed in previous examples, optimal covalent bindingwas at 50% VDM.

EXAMPLE 9 The Time-Course for the Binding of Protein A to VDM-GraftedMembranes

Table 6 summarizes the results of several previous experiments inbinding Protein A to grafted PE membranes in which length of incubationof the protein with the membrane was varied. Although the experimentalconditions were not exactly comparable, they showed a trend that proteinbinding is highest if allowed to proceed overnight (16 h). Anapproximation of overnight binding can be obtained within one workday byincubating for at least 5.5 h.

                  TABLE 6                                                         ______________________________________                                        Time-Dependence of the Binding of Protein A to                                VDM-Grafted Membrane                                                                                Protein A                                               Example      Time (h) Bound (μg/cm.sup.2)                                  ______________________________________                                        4            1        3.2                                                     7            5.5      5.6                                                     6            16       6.2                                                     5            16       7.0                                                     ______________________________________                                    

These results, obtained from passive diffusion of protein into themembrane, demonstrated a definite time-dependence. A dramaticacceleration (perhaps a hundred-fold) would occur if protein were drawninto the membrane through some active process such as slight pressuredifferential.

EXAMPLE 10 The Effect of High Salt Concentration on Binding Protein A

Using the teachings, specifically Examples 1-35 of coassigned, copendingU.S. patent application Ser. No. 07/609,436 (Coleman et al.),incorporated herein by reference (also published as PCT Publication WO92/07879), it was investigated whether high sulfate concentration couldalso enhance the binding of protein to azlactone which had been E-beamgrafted to PE membranes.

Protein A was incubated with various membranes for 19 h in 25 mM sodiumphosphate, pH 7.5, 150 mM NaCl (low salt). In the "high salt" incubation1.5M sodium sulfate was substituted for the sodium chloride. Controlswere e-beam- and solvent-treated.

                  TABLE 7                                                         ______________________________________                                        The Effect of Sodium Sulfate on Protein                                       Coupling to Grafted Membranes                                                              Ratio of "High Salt" to                                                       "Low Salt" Binding                                                              Total                                                                         Protein             Coupled                                                   Bound     SDS       Protein                                    Membrane       (μg/cm.sup.2)                                                                        Resist. (%)                                                                             (μg/cm.sup.2)                           ______________________________________                                        Control        9.5/5.3   30/20     2.8/1.0                                    Control + 25% VDM                                                                            10.5/5.8  45/30     4.8/1.8                                    Control + 50% VDM                                                                            9.4/4.9   55/34     5.1/1.6                                    Control + 100% VDM                                                                           6.6/2.7   70/58     4.6/1.5                                    ______________________________________                                    

The results are quite consistent with those observed with Protein A onhydrophilic azlactone-functionalized porous polymeric beads as shown inExamples 1-35 of U.S. Pat. No. 5,200,471. There was 2.5-3-fold increasein the amount of coupled protein, a 75-150% increase in total binding,and an increase in SDS resistance of 20-50%. Additionally theseexperiments confirmed conclusions drawn in earlier experiments: optimalVDM concentration is less than 100% and perhaps about 50%; thepercentage of the binding which is SDS resistant continued to increasein proportion to the percentage of azlactone.

EXAMPLE 11 Azlactone-Grafted Membranes are Useful in an Immunodiagnostic

One of the major uses of specialized, biocompatible membranes inbiotechnology is to immobilize one of the reactants in a clinicaldiagnostic test as in a heterogeneous ELISA-type assay. See for example,European Patent Publication 0 294 105 (Rothman et al.). In this example,it was demonstrated that an azlactone-functionalized membrane could beused to bind an antibody and that the resulting derivatized membranecould be used in a chromogenic ELISA (enzyme linked immunosorbentassay).

Strips of azlactone-functionalized membranes prepared according toExample 3 above were incubated with continuous rocking for 17 h atambient temperature with either human IgG (hIgG in 100 mM NaCl and 100mM sodium phosphate solution, pH 7.25) or bovine serum albumin (BSA),each at 1.0 mg/ml, in 10 ml of 25 mM sodium phosphate, 150 mM NaCl, pH7.5. They were then given several 1 h rinses in buffer, dried, andstored, desiccated, at ambient temperature until used. Prior to use, toinsure that all azlactone-functional moieties were reacted, membranediscs were incubated with 3.0M ethanolamine and 1 mg/ml BSA, pH 9.0, for30 min., rinsed and used in the assay described below.

The assay was initiated by incubation of the discs with 10 ug/mlanti-human IgG-HRP (horseradish peroxidase) conjugate(Cappel-Worthington, Malvern, Pa.) for 1 h with continuous rocking. Theywere rinsed for 4 h, with rocking, with PBS-Tween (25 mM sodiumphosphate, 0.6% Tween 20, pH 7.5) and transferred to clean test tubesfor a chromogenic HRP substrate, o-phenylenediamine (Sigma Chem. Co.) (3mM in 100 mM sodium citrate buffer, 0.12 mg 30% hydrogen peroxide, pH5.0). The product of the peroxidation forms an orange-colored,partially-insoluble product after reaction with 2.5M H₂ SO₄.Spectrophotometric estimations of the reaction were obtained bytransferring 50 μl of the reaction supernate to a 96-well microtiterplate containing 20 μl of 2.5M H₂ SO₄. Results of absorbancedeterminations at 490 nm on a microtiter plate spectrophotometer(Dynatech, Chantilly, Va.) are given in Table 8.

                  TABLE 8                                                         ______________________________________                                        Comparison of the Binding of Anti-IgG-HRP to                                  Control and IgG-Containing Membranes                                                  HRP Activity (mA @ 490/min)                                                                     IgG/BSA                                             Sample    BSA-Treated IgG-Treated Ratio                                       ______________________________________                                        25% VDM   98          451         4.9                                         50% VDM   60          201         3.4                                         100% VDM  41          199         5.5                                         ______________________________________                                    

In each case there is considerably more activity associated with theantibody-containing membranes than with the BSA controls.

In this example a 150,000 dalton antibody was immobilized, thencomplexed with a 200,000 dalton antibody-enzyme conjugate, indicatingthat there is not a great barrier to working with large proteincomplexes.

EXAMPLE 12 Gamma Irradiation Grafting of Hydrophilized MicroporousMembrane with VDM and HEMA

15 preweighed pieces of PE microporous membrane, having a thin shell ofpoly(vinyl alcohol) prepared according to Example 22 of coassigned,copending application Ser. No. 08/122,807 (Gagnon et al.) and PCTPublication WO 92/07899, except that PE was used instead of PP,measuring 7.6×20.3 cm, were rolled-up and placed into glass ampules. Theampules were evacuated to pressures less than 2×10⁻⁴ mm Hg and the glassnecks were melt-sealed to prevent O₂ contamination. Three additionalpieces were placed in unsealed test tubes. All 18 sample tubes wereexposed to gamma irradiation for 9.5 hours for a total dose of 38 kGys.The tubes had been placed side-by-side in a large envelope which wasconfigured normal to the source, so that they would all be exposed tothe same dose. The envelope was rotated 180° after about the first 4hours of irradiation to further ensure that the samples were dosedevenly.

After gamma irradiation, the tubes were placed into a glove bag, whichalso contained the argon-purged (i.e., O₂ -free) monomer solutionslisted in Table 9 below. The glove bag was flushed with argon by 5inflate/deflate cycles to remove as much O₂ as possible. Four sealedampules were broken open, and the film samples within them were placedin either pure ethyl acetate (EtOAc), 10 wt/vol % VDM in EtOAc, or 10%VDM/25% HEMA in EtOAc, respectively, These were allowed to soak forlonger than 5 minutes, before being removed from the solution and storedin stoppered test tubes. A total of three samples were prepared for eachmonomer solution. After all reactions were complete, the samples wereremoved from the glove bag and rinsed 3 times in fresh EtOAc to removeexcess monomer, and air dried.

The membranes were analyzed for grafting add-on by weight uptake and byFT-IR spectroscopy. The weight uptake data showed that the 10% VDM and10% VDM/25% HEMA membranes averaged about 0.7 and 1.0% wt. uptake. TheIR spectra confirmed the wt. uptake data, showing significant absorbanceat 1824 cm⁻¹ for VDM in both the 10% VDM and 10/25 VDM/HEMA samples. Thesamples also displayed an absorbance at 1670 cm⁻¹, indicative of partialhydrolysis of the VDM moiety. An additional absorption peak at 1728 cm⁻¹for the 10/25 VDM/HEMA samples confirmed the incorporation of the HEMAmonomer into the grafted copolymer. No 1728 cm⁻¹ peak could be seen inthe 25% HEMA membranes. The wt. uptake data and the absorbance valuesfor the VDM and HEMA functionalities, normalized to the PE absorbancepeak at 1462 cm⁻¹, are tabulated below:

                  TABLE 9                                                         ______________________________________                                                         IR ABSORB-                                                                    ANCE RATIO                                                              AVE WT %    1824 cm.sup.-1 /                                                                         1728 cm.sup.-1 /                            SAMPLE     ADD-ON      1471 cm.sup.-1                                                                           1471 cm.sup.-1                              ______________________________________                                        Control    0.000       0.000      0.000                                       γ only                                                                             0.115       0.000      0.000                                       γ in air                                                                           0.160       0.000      0.000                                       γ + EtOAc                                                                          0.160       0.000      0.000                                       γ + 10% VDM                                                                        0.718       0.060      0.000                                       γ + 10 VDM/                                                                        1.013       0.040      0.120                                       25 HEMA                                                                       ______________________________________                                    

Summing the IR absorbance ratios (including the 1670 cm⁻¹ peak) gives anindication of overall add-on.

EXAMPLE 13 E-beam Irradiation Grafting Hydrophilized MicroporousMembrane with VDM and HEMA

Pre-irradiation e-beam grafting of hydrophilized PE microporousmembranes, prepared according to Example 3 above used the same equipmentof Example 1 above, except that modifications were made to the glove boxto minimize presence of O₂. An O₂ analyzer, installed in the glove boxto monitor the O₂ concentration during the run, showed that theseimprovements allowed the O₂ concentration to be maintained at less than30 ppm--often as low as 10 ppm.

Pieces of the membrane were taped to a polyethylene terephthalate (PET)carrier web and passed through an e-beam curtain at 6.1 m/min. Thee-beam accelerating voltage was set at 150 KeV, and a dose rate of 50kGys was used to irradiate the membranes. The samples came out of thee-beam chamber directly into a N₂ purged glove box where they wereimmersed in the monomer solution. The inert atmosphere helped to preventquenching of the generated radicals with oxygen.

The solutions had concentrations (in wt %) VDM and HEMA in ethyl acetatein concentrations listed in Table 10 below.

Irradiated membrane samples were soaked in the monomer solution for 24hours, followed by a three 5 minute soaks in fresh ethyl acetate inorder to wash out excess monomer. They were then dried and placed inziplock type bags to prevent possible hydrolysis of the azlactone byatmospheric water.

The membranes were incubated for 16 h with Protein A at 250 μg/ml ineither 25 mM or 500 mM sodium phosphate buffer (pH 7.50) which wassupplemented with 150 mM NaCl or 1.5M sodium sulfate. The results arepresented in Table 10. Those experiments performed in the 25 MM bufferare indicated by an asterisk. All results are the averages oftriplicates.

                  TABLE 10                                                        ______________________________________                                        Effects of Grafting HEMA in Combination with                                  VDM into PE Membranes                                                                     Initial    SDS        Coupled                                     Sample      Binding    Resistance Binding                                     (% VDM/HEMA)                                                                              (μg/cm.sup.2)                                                                         (%)        (μg/cm.sup.2)                            Salt        Cl  SO.sub.4                                                                             Cl SO.sub.4                                                                              Cl  SO.sub.4                                ______________________________________                                        Untreated   10.66/9.78  8/10      0.81/0.97                                   Solvent     11.57/10.29                                                                              21/31      2.38/3.18                                   E-beam*     11.97/9.52 23/30      2.72/2.86                                   E-beam      10.59/10.13                                                                              23/31      2.44/3.09                                   0/2.5*      0.43/2.02  27/36      0.11/0.73                                   0/6.5*      0.35/1.41  28/34      0.10/0.49                                   0/10        0.27/1.81  24/42      0.06/0.74                                   0/12.5      0.80/3.85  25/38      0.21/1.45                                   0/25*       0.30/1.07  28/32      0.09/0.36                                   0/25        0.26/1.01  24/38      0.06/0.38                                   10/0*       4.06/5.14  29/36      1.19/1.85                                   10/0        4.33/7.21  29/42      1.25/3.05                                   25/0*       2.68/3.58  36/42      0.95/1.47                                   25/0        2.73/4.30  38/49      1.05/2.11                                   50/0        2.78/3.65  77/84      2.14/3.06                                   10/10       3.57/8.96  56/81      2.01/7.28                                   10/25*      2.10/9.70  67/94      1.52/9.10                                   10/25        0.82/15.55                                                                              60/95       0.49/14.72                                 25/2.5*     2.45/4.53  54/74      1.33/3.35                                   25/6.25*    2.69/5.78  82/89      2.14/5.11                                   25/12.5     5.10/8.98  51/78      2.62/7.01                                   25/25       1.48/9.82  63/92      0.93/9.04                                   50/10       1.86/3.30  69/77      1.29/2.56                                   ______________________________________                                    

EXAMPLE 14 UV Initiated VDM Grafting on a Hydrophilized, MicroporousMembrane

A piece of hydrophilic PE membrane prepared in the same manner as forExample 12 above was soaked with an ethyl acetate solution of 25 wt/vol% VDM in ethyl acetate +0.25% uv initiator azobis(isobutyronitrile)(commercially available as Irgacure™ 907 from Ciba Geigy) and then fedinto a N₂ purged RPL uv treater at 7.6 m/min. set at 21 amp lamp power(310 kW/m²). Another sample was treated the same way, except that themonomer solution also contained 2.5 wt % crosslinker (neopentylglycoldiacrylate, NPGDA). Some evaporation of the monomer solution did occurprior to, and during the irradiation.

IR spectroscopy showed that VDM did indeed become grafted onto themembrane surfaces in both cases. Using the ratio of the IR absorbancefor VDM at 1824 cm⁻¹ to the absorbance of PE at 1462 cm⁻¹ as a measureof VDM add-on, showed that the sample without the NPGDA had a greateradd-on than that with the crosslinker.

EXAMPLE 15 Pre-Irradiation EB Grafting onto H-PP Membrane and PE BMF

Hydrophilic polypropylene (PP) membrane was prepared in the manneraccording to Example 22 of coassigned, copending U.S. patent applicationSer. No. 07/775,969 (Gagnon et al.) published as PCT Publication WO92/07899. PE blown microfiber (BMF) web was prepared according toExample 19 of Gagnon et al.),to become a calendared BMF nonwoven madefrom Dow 6808 LLDPE resin at a basis wt. of 94 g/m².

All samples of PP membrane and PE BMF nonwoven were irradiated in themanner according to Example 13 above with 50 kGys of 150 KeV e-beamirradiation prior to immersion in the monomer solutions listed in Table11 in a <30 ppm 02 atmosphere. All monomer solutions, in ethyl acetate,had been purged with argon to remove O₂. Reaction was allowed to proceedfor about 5 minutes prior to removal and rinsing in pure ethyl acetate.

The table below lists the grafting wt. % add-on, expressed as ##EQU1##for the samples in terms of the monomer solution used.

                  TABLE 11                                                        ______________________________________                                        E-beam Grafted VDM on PP Membrane and PE BMF                                               WEIGHT PERCENT ADD-ON                                                          PP-Membrane                                                                             PE-BMF                                                ______________________________________                                        10% VDM          23%         15%                                              25% HEMA        265%        328%                                              10/25 VDM/HEMA  215%        136%                                              ______________________________________                                    

Infrared spectroscopy confirmed that these monomers were indeedincorporated onto the surfaces of these substrates as grafted polymers.

EXAMPLE 16 Preparation of and Protein Binding of Plasma VDM-TreatedMaterials

Hydrophilized, porous polyethylene (PE) membrane prepared according toExample 12 above was used without further treatment. Non-porous films ofpolypropylene (biaxially oriented, thermally extruded, 0.05 mm thick PPfilm), poly(ethyleneterephthalate) (biaxially oriented, 0.1 mm thick,PET film) and poly(tetrafluoroethylene) (0.05 mm thick PTFE film) wereused without additional treatment.

Vinyldimethyl azlactone (VDM) was deposited onto all the film samplessimultaneously in a glow discharge. The glow discharge depositionsoccurred in a belljar vacuum system using two parallel plate electrodes(20 cm×30 cm) spaced 5 cm apart. The film samples were placed on thelower electrode (grounded). The materials were subjected to a VDM glowdischarge at 60 mtorr VDM pressure with a 15 W discharge power generatedat a frequency of 25 kHz. First one side was treated, then the sampleswere turned over on the bottom electrode to treat the other side. Thenominal thickness of the deposition was 70 nm on each side of thesamples, as measured by a quartz-crystal-microbalance exposed to thedischarge during the deposition.

Alternatively, the film samples were given an initialnitrogen-containing surface by nitrogen discharge prior to VDMdeposition (noted in Table 12 below as "N/VDM" treatment). Prior to glowdischarge deposition the samples were treated with nitrogen gas (200mtorr) glow discharge of 15 W (25 kHz) for 10 s. This was followed bythe VDM treatment as described above.

Triplicate (8 mm) discs of each material were cut using a standardoffice paper punch and placed in 2 mL micro centrifuge tubes followed byaddition of 200 μL of the buffer solution containing radioiodinatedProtein A (ranging from 2200 to 2500 cpm/ug of protein). The chloridebuffer consisted of 150 mM NaCl and 500 mM sodium phosphate, pH 7.5; thesulfate buffer was 1.5M sodium sulfate and 500 mM sodium phosphate, pH7.5. The discs were incubated with the solutions for 17 h (withcontinuous rocking) to allow the protein to fully equilibrate throughoutthe porous membrane. The coupling reaction was stopped by addition of500 uL of 1.0M ethanolamine (in 25 mM sodium pyrophosphate, pH 9.0),twice for a total of 5 h. After three additional washes with thechloride buffer the discs were transferred to another test tube, and theassociated radioactivity was determined using a Packard Model 5230 gammaradiation detector.

Protein which was resistant to solubilization by a treatment with theprotein denaturant sodium dodecyl sulfate (SDS) was operationallydefined as "covalently coupled" to the substrate. This treatment waswith a 1% SDS solution (in 25 mM sodium phosphate buffer, pH 7.5) for 4h at 37° C., followed by three washings with the warm SDS solution, andredetermination of the amount of associated radioactivity.

                  TABLE 12                                                        ______________________________________                                        The Coupling of Protein to Plasma-Treated                                     Materials                                                                                        Protein            Protein                                                    Binding   SDS      Coupling                                                   (μg/cm.sup.2)                                                                        Resistance                                                                             (μg/cm.sup.2)                        Material                                                                              Treatment  Cl/SO.sub.4                                                                             (%)      Cl/SO.sub.4                             ______________________________________                                        PE      Control    11.2/9.5  33/34    3.7/3.2                                         VDM         7.6/12.0 68/75    5.2/9.0                                         N/VDM       7.9/20.6 79/83     6.2/17.0                               PP      Control    0.6/0.4   21/17    0.1/0.1                                         VDM        0.2/0.5   42/54    0.1/0.3                                         N/VDM      0.9/0.7   75/69    0.7/0.5                                 PET     Control    1.4/0.8   27/20    0.4/0.2                                         VDM        0.5/1.4   55/70    0.3/1.0                                         N/VDM      0.4/1.1   62/65    0.2/0.7                                 PTFE    Control    0.4/0.3   22/17    0.1/0.1                                         VDM        0.2/0.7   38/72    0.1/0.5                                         N/VDM      0.3/1.2   56/76    0.2/0.9                                 ______________________________________                                    

Comparing just the Control samples, much more Protein A binds to the PEmembrane than to any of the films. This is understandable because themembrane has about ten-fold more total surface area than the films.Thus, it is quite surprising to observe a two-fold increase in theinitial binding resulting from the nitrogen/VDM treatment, since thenitrogen treatment and VDM deposition do not penetrate substantiallyinto the pores of the membrane. See Example 19 below. Actual enhancementof protein binding to PE is closer to the 400% observed for the PTFEfilms, than the 30% seen with the PET films. Such a high enhancementfactor on PE membranes means that one might make a single-layer membraneact like a laminated, multi-layered membrane by surface treatment on oneside to produce layer A, followed by treatment on the other side toproduce layer C, followed by treatment with an e-beam or otherpenetrating activator to produce layer B. Layers A, B, and C mightrepresent three different grafted monomers, conferring, e.g., differenthydrophilicities or wettabilities, etc., or, perhaps, the three layerswould use the same monomer, e.g., VDM, with which three differentproteins or other ligands were immobilized to make, for example, asimple-to-use immunodiagnostic device.

EXAMPLE 17 Preparation of Corona-Treated Azlactone-Functional Supports

The corona deposition of samples (prepared according to Example 16) wascarried out in a belljar system using two metal rollers (10 cm diameter,15 cm long) for electrodes. The grounded electrode was covered with a 2mm thick sleeve of silicone rubber and the electrodes were separated bya 1.7 mm gap. The samples were mounted on the silicone sleeve usingtape. The rollers rotated at 25 rpm causing the samples to be repeatedlyexposed to the discharge in the region of closest proximity between thetwo rollers. The belljar was evacuated to remove the air atmosphere andbackfilled with 100 mtorr VDM and He to a pressure of 1 atm. The sampleswere exposed to a 250 W corona discharge (40 kHz) for 3 minutes ofrotation (approximately 30 s actual exposure to the discharge).

Alternatively, similar to that described in Example 16, the samples weresubjected to a nitrogen gas corona treatment (1 atm) of 250 W (40 kHz)for 15 s of rotation (2.5 s of exposure). This was followed by VDMtreatment as described above. These samples are indicated in Table 13 asthe N/VDM treatment.

The protein binding experiments were performed identically to thosedescribed in Example 16 except that the specific radioactivity of theProtein A was 1300-1700 cpm/μg of protein.

                  TABLE 13                                                        ______________________________________                                        The Coupling of Protein to Corona-Treated                                     Materials                                                                                        Protein            Protein                                                    Binding   SDS      Coupling                                                   (μg/cm.sup.2)                                                                        Resistance                                                                             (μg/cm.sup.2)                        Material                                                                              Treatment  Cl/SO.sub.4                                                                             (%)      Cl/SO.sub.4                             ______________________________________                                        PE      Control    13.8/10.5 35/32    4.8/3.3                                         VDM        14.8/23.0 81/86    12.0/19.8                                       N/VDM      12.7/8.8  78/77    9.9/6.9                                 PP      Control    1.2/0.4   15/15    0.2/0.1                                         VDM        1.9/1.0   58/40    1.1/0.4                                         N/VDM      2.0/1.0   66/45    1.4/0.5                                 PET     Control    1.8/0.8   24/19    0.4/0.2                                         VDM        1.9/0.9   49/36    0.9/0.3                                         N/VDM      1.3/0.8   62/44    0.8/0.3                                 PTFE    Control    0.6/0.4   17/15    0.1/0.1                                         VDM        1.5/0.8   70/57    1.0/0.5                                         N/VDM      1.3/1.0   72/64    1.0/0.6                                 ______________________________________                                    

Corona treatment yields results very similar to those observed inExample 16 with plasma treatment. There are differences in the absolutevalues of some of the numbers, but the general affect is the same:addition of azlactone functionality to the surface results in anincrease in the amount of coupled protein.

In the event that it were desired to graft azlactone-functionality tointerior surfaces of a porous, pre-existing support, one could shieldboth electrodes with silicone rubber sleeves (like that described above)and employ the same corona discharge procedure using helium as describedin this Example to achieve a penetrating VDM treatment.

EXAMPLE 18 The Preparation of and Protein Binding to Plasma VDM-TreatedPorous and Fibrous Substrates

VDM was deposited onto the following materials in a glow dischargeprocedure as described in Example 16:

PP blown microfiber web (basis weight of 60 g/m² and fiber diameter of5-10 μm) prepared by melt-blowing techniques disclosed in van Wente etal. "Superfine Thermoplastic Fibers" Industrial Engineering Chemistry,Vol. 48, pages 1342 et seq. (1956) and van Wente et al "Manufacture ofSuperfine Organic Fibers" Report No. 4364 of Naval Research Laboratories(May 25, 1954);

Celgard™ microporous polypropylene membrane 2402 (commercially availablefrom Hoechst-Celanese, Charlotte, N.C.);

Polyurethane commercially available from Dow Chemical under thetradename "Pellethane 2363-65D".

Rayon blown microfiber web, such as that used in Micropore™ tape,commercially available from Minnesota Mining and Manufacturing Company;and

The PP film and hydrophilized porous PE membrane treated in the mannerdescribed in Example 16.

The non-porous PP film was also subjected to simultaneous treatment witha combination of VDM and hydroxyethyl methacrylate (HEMA) using a glowdischarge (50 mtorr VDM, 10 mtorr HEMA, 15 W) to deposit a 70 nmcoating.

Protein binding experiments were performed as described in Example 16,except that the blown microfiber web and Celgard materials required 0.2%Triton X-100 in order to be thoroughly wetted by the buffer solutions.

The specific radioactivities varied from 5100 to 6500 cpm/μg of protein.The ethanolamine quenching steps were for a total of 3 h.

                  TABLE 14                                                        ______________________________________                                        The Coupling of Protein to Plasma-Treated                                     Materials                                                                                        Protein           Protein                                                     Binding   SDS     Coupling                                                    (μg/cm.sup.2)                                                                        Resistance                                                                            (μg/cm.sup.2)                         Material                                                                             Treatment   Cl/SO.sub.4                                                                             (%)     Cl/SO.sub.4                              ______________________________________                                        PP     Control     0.37/0.26 6/9     0.02/0.02                                       VDM         0.17/0.77 23/76   0.04/0.58                                       VDM/HEMA    0.16/0.79 23/80   0.04/0.63                                PE     Control     3.50/6.48 45/62   1.57/4.00                                       VDM          1.77/16.10                                                                             86/91    1.54/14.71                              Rayon  Control     0.06/0.17 26/40   0.02/0.07                                       VDM         0.08/1.33 34/91   0.03/1.22                                PU     Control     0.30/0.33 29/38   0.09/0.12                                       VDM         0.19/0.68 47/64   0.09/0.44                                PP/    Control     0.03/0.27 13/15   0.004/0.04                               BMF*   VDM         0.03/3.91 25/93   0.009/3.65                               Cel-   Control     0.03/0.93  9/18   0.003/0.17                               gard*  VDM         0.04/1.10 19/68   0.007/0.75                               ______________________________________                                         *indicated Triton X100 in protein incubation solution                    

Azlactone-modification of rayon (a cellulose-based synthetic polymer)and PU yield 17-fold and four-fold protein coupling increases,respectively. The PP microfiber shows a tremendous hundred-fold increaseupon azlactone modification. The modification of Celgard polyethylene isespecially useful because Celgard polyethylene is often a material usedto make microporous hollow fiber filtration membranes. Addition of apleotropic agent such as azlactone to hollow fiber membranes wouldincrease their utility many fold.

COMPARISON EXAMPLE 19 Glow Discharge Treatment Does Not Penetrate intoPorous Material

Two 10 cm×10 cm pieces of microporous PE membrane, 20 μm thick, preparedaccording to Example 23 of U.S. Pat. No. 4,539,256 (Shipman) were tapedtogether along their edges and placed on the bottom electrode for plasmaglow discharge treatment in the belljar vacuum system according toExample 16. The membrane was treated with a glow discharge of VDM (60mTorr), as described in Example 16. Two samples were prepared, one witha 70 nm coating and the other with a 150 nm coating of the VDM-plasmapolymer. After treatment, the upper layer of the two-layer constructionwas separated from the lower layer, and analyzed by X-ray photoelectronspectroscopy (XPS). Both surfaces of this membrane, the top surface(exposed to the electrical discharge) and the bottom surface (which wasin contact with the lower membrane), were analyzed.

                  TABLE 15                                                        ______________________________________                                        XPS Analysis of the External Surfaces of                                      Discharge-Treated 20 μm Porous Membranes                                                    Atomic Ratios                                                Sample             O/C    N/C                                                 ______________________________________                                        70 nm - top        0.23   0.12                                                70 nm - bottom     0.0    0.0                                                 150 nm - top       0.22   0.12                                                150 nm - bottom    0.0    0.0                                                 ______________________________________                                    

The top surfaces clearly have azlactone-functionality, as evidenced bythe oxygen and nitrogen atom content. The bottom surfaces of themembranes are untreated PE, with no oxygen or nitrogen present. Thisdemonstrated that the electrical-discharge-deposited polymer of VDM doesnot penetrate appreciably into the pores of the membrane, even a verythin membrane and even when very heavily loaded with VDM (as evidencedby the ratios of O and N to C which are very near the theoretical valuesexpected for an "infinitely thick" layer of pure VDM, 0.29 and 0.14,respectively). This experiment demonstrated the feasibility of the"multilayered" single membrane devices discussed in Example 16.

EXAMPLES 20-51 Crosslinked Azlactone-functional Coatings on NonwovenPolymeric Supports

Table 16 below shows the results of a series of experiments to preparecrosslinked azlactone-functional coatings on surfaces of nonwovenpolymeric supports. The methods of preparation follow Table 16.

                                      TABLE 16                                    __________________________________________________________________________    Azlactone - Functional Coatings                                               COATED NONWOVENS TABLE                                                                                           COUPLED                                                                  % SDS                                                                              PROTEIN A                                    MONOMER FORMULATION                                                                           NONWOVEN    RES. μg/cm.sup.2                             __________________________________________________________________________    20                                                                              20:20:60        REEMAY-2    93   21.17                                      21                                                                              EGDMA/VDM/HEMA  REEMAY-1    68   1.45                                       22                                                                              (50 g in 400 mL IPA)                                                                          PET TB      86   11.04                                      23                PET LB      82   7.70                                       24                PP OE       53   1.77                                       25                PUR         74   3.02                                       26                COTTON      89   20.53                                      27                                                                              80:20 TMPTMA/VDM                                                                              CEREX       75   3.70                                       28                                                                              50 g in 400 mL Hexanes                                                                        PET CW      65   2.91                                       29                DUPONT SONTARA                                                                            85   9.53                                       30                PE/PP-3     64   65.20                                      31                PE/PP-10    37   16.66                                      32                                                                              70:20:10        DUPONT SONTARA                                                                            68   1.43                                       33                                                                              BA/VDM/TMPTMA   CFX NYLON   70   1.79                                       34                                                                              (50 g in 400 mL PP          52   0.29                                       35                                                                              Hexanes)        RAYON       73   2.06                                       36                PP NP       26   1.07                                       37                                                                              70:20:10        DUPONT SONTARA                                                                            81   2.71                                       38                                                                              BA/VDM/EGDMA    CFX NYLON   69   2.09                                       39                                                                              (50 g in 400 mL PP          33   0.27                                       40                                                                              Hexanes         RAYON       77   9.92                                       41                PP NP        4   0.23                                       42                                                                              70:20:10 IOA/VDM/EGDMA                                                                        DUPONT SONTARA                                                                            66   1.38                                       43                                                                              (500 g in 400 mL Hexanes)                                                                     CFX NYLON   72   1.37                                       44                PP          36   0.21                                       45                RAYON       75   1.28                                       46                PP NP       32   1.04                                       47                                                                              50:20:20:10     DUPONT SONTARA                                                                            74   1.19                                       48                                                                              IBMA/BMA/VDM/TMPTMA                                                                           CFX NYLON   63   1.27                                       49                                                                              (50 g in 400 mL Hexanes)                                                                      PP          49   0.30                                       50                RAYON       76   2.49                                       51                PP NP       34   2.37                                       __________________________________________________________________________    PROTEIN A   protein A coupled using 1.5M sulfate in 0.2M sodium                           phosphate                                                                     buffer                                                            REEMAY-1    Style 2200 spunbonded polyester from REEMAY of Old                            Hickory, Tennessee                                                REEMAY-2    Style 2295 spunbonded polyester from REEMAY                       DUPONT SONTARA                                                                            Rayon/polyester (basis weight 135 g/m.sup.2 from DuPont)          CEREX       Type 23 Nylon 66 (basis weight 34 g/m.sup.2) from Fiberweb                    of                                                                            Simpsonville, S.C.                                                PET TB      Thermal bonded polyester prepared from CELLBOND                               branded bicomponent fiber (25μ fiber diameter) on air laid                 web                                                                           former from Rando Machine and using air circulation oven                      for binding                                                       PET LB      Air laid polyester bonded with Rohm and Haas branded latex        PP OE       Oriented, embossed polypropylene (8μ fiber diameter)melt                   blown according to U.S. Pat. No. 4,988,560 (Meyer et al.)         PUR         Melt blown polyurethane (8μ fiber diameter) melt blown                     according to Wente et al. "Superfine Thermoplastic Fibers"                    in Industrial Engineering Chemistry, Vol. 48, page 1342 et                    seq. (1956)                                                       COTTON      Spunlaced cotton from Veratec Corporation                         PET CW      Mechanically laid, embossed polyester having 2 denier fiber       PE/PP-3     Needlepunched, air laid polyethylene sheathed polyproyene                     having 3 denier fiber                                             PE/PP-10    Needle punched, air laid polyethylene sheathed polypropylene                  having 10 denier fiber                                            CFS Nylon   Melt blown CFX nylon copolymer from Allied                        PP NP       Needlepunched, air laid polypropylene having 205 g/m.sup.2                    basis                                                                         weight                                                            PP          Air laid polypropylene having 205 g/m.sup.2 basis weight          RAYON       Needlepunched, air laid rayon having 135 g/m.sup.2 basis                      weight                                                        

EXAMPLES 20 20:20:60 EGDMA/VDM/HEMA Coating on Spunbonded Polyester

A spunbonded polyester sample (REEMAY, style 2200; 15 cm square) wasdipped into a 2-propanol solution of 20 parts EGDMA, 20 parts VDM and 60parts HEMA (prepared by dissolving 10.0 g EGDMA, 10.0 g VDM, 30.0 gHEMA, and 1.0 g Darocure 1173 in 400 ml 2-propanol), and then pressedbetween sheets of polyethylene to remove excess solution. After purgingthe sample with N₂ for 3 minutes, the monomer coating was polymerized byexposure, under N₂, to low intensity UV irradiation for 12 minutes. Thesupport was then soaked in 2-propanol for 1 minute and air dried.Analysis by attenuated total reflectance IR (ATIR) revealed thecharacteristic azlactone carbonyl absorption at 1820 cm⁻¹ indicatingazlactone incorporation in the polymer coating. SEM analysis of thetreated polyester revealed no particulates and indicated a uniformcoating of the fibers. The azlactone-functional spunbonded polyestersupport coupled 21.17 μg of radiolabeled Protein A per cm² (measuredafter SDS treatment).

EXAMPLES 21, 23-26

The nonwoven samples 21 and 23-26 were coated and cured in the samemanner as Example 20 except a different nonwoven was employed, asidentified in Table 16.

EXAMPLE 22 20:20:60 EGDMA/VDM/HEMA Coating on Thermal Bonded Polyester

The thermal bonded polyester sample (25 μm thick, 15 cm square) wascoated and cured in the manner of Example 1. Analysis of the finishedsupport by ATIR revealed the azlactone carbonyl absorption at 1820 CM⁻¹.SEM analysis revealed the coating to be grainy and particulate. Thethermal bonded polyester azlactone-functional support coupled 11.04 μgof radiolabeled Protein A per cm² (measured after SDS treatment).

EXAMPLE 30 80:20 TMPTMA/VDM Coating on Polypropylene

A polypropylene sample (3 denier, needle punched, 15 cm square) wasdipped into a hexane solution of 80:20 TMPTMA/VDM (prepared bydissolving 40.0 g TMPTMA, 10.0 g VDM, and 1.0 g Darocure 1173 in 400 mLhexane). Excess solution was removed from the web by pressing it betweensheets of polyethylene. After purging with N₂ for 3 minutes, the monomercoating was polymerized by exposure under N₂, to low intensity UVirradiation for 13 minutes. Because of the thickness of this sample, theweb was flipped over, purged, and irradiated for an additional 6minutes. The azlactone-functional support was then rinsed with hexaneand air-dried. SEM analysis of the composite revealed a grainyparticulate coating with some agglomerated particles. The Daiwacomposite coupled 65.2 μg of radiolabled Protein A per cm² (measuredafter SDS treatment).

EXAMPLES 27-29 AND 31

The nonwoven samples of Examples 27-29 and 31 (15 cm square) were coatedand cured in the manner of Example 30, except that Examples 27-29 wereirradiated on one side only for 8-11 min. and Example 31 was irradiatedon both sides in succession for 7 min each.

EXAMPLES 32-36

The nonwoven samples of Examples 32-36 were dipped into a hexanessolution of 70 parts BA, 20 parts VDM, and 10 parts of TMPTMA, (preparedby dissolving 35.0 g BA, 10.0 g VDM, 5.0 g TMPTMA, and 1.0 g Darocure1173 photoinitiator in 400 ml of hexanes) and then were pressed betweensheets of polyethylene to remove excess solution. After purging with N₂for 3 min., the monomer coating was polymerized by exposure under N₂, tolow intensity uv radiation for 10 mins. The azlactone-functionalsupports were soaked and rinsed with hexanes and then dried under N₂.

EXAMPLES 37-46

The nonwoven samples of Examples 37-46 (10 cm square) were coated with ahexanes solution of 70 parts BA, 20 parts VDM, 10 parts EGDMA,containing 2% Darocure™ 1173 photoinitiator and then cured in the mannerof Examples 32-36.

EXAMPLES 47-51

The nonwoven samples of Examples 47-51 (10 cm square) were coated with ahexanes solution of 50 parts IBMA, 20 parts BMA, 20 parts VDM, 10 partsTMPTMA, containing 2% Darocure™ 1173 and then cured with 12 minutes ofirradiation in the same manner as Examples 32-36.

EXAMPLE 52 Preparation of Azlactone-Functional Polymethylmethacrylate

Poly(methylmethacrylate) (PMMA commercially available as Perspex CQ, UVfrom ICI) buttons were exposed to one of several electron beam dosages:grafting at conditions of 10, 20, 30, 50, or 100 kGys discharged at 100kV in N₂ gas a flow of less than 4 scm/min, at a line speed of 9.2m/min, with O₂ content less than 50 ppm. The activated PMMA substratewas immediately transferred into VDM monomer for grafting. The graftingwas immediately started on the surface of the support. The buttons werewashed with VDM monomer and anhydrous ethyl-ether. When the activatedsupport was immersed longer (e.g., several hours), in VDM monomer, theVDM-grafted PMMA was dissolved in the VDM monomer. Therefore, tominimize dissolution, a heptane/VDM (75/25 wt. %) solution wassubstituted for VDM monomer. It was found that the surface of the buttonsupport remained intact. As a control, the PMMA buttons were exposed toelectron beam only to see how much polymeric chains were degraded byelectron beam. Evidence of VDM grafting on the PMMA button was confirmedby ATR-FTIR, ¹³ C NMR and XPS (ESCA). The molecular weight distributioncurve indicated there was no degradation of the polymeric chains of thesupport at all under the conditions of 10 kGys electron beam dose.

EXAMPLE 53 Azlactone-functional PMMA Prepared and Reacted withAmine-Terminated Heparin.

PMMA casted film (Perspex, CQ, UV; <0.1 mm thickness) was exposed to theelectron beam in a N₂ blanket, and then conveyed into the glove boxunder N₂ gas where the activated film was immersed in VDM monomer fortwelve hours at room temperature. The film lost all of its originalshape but was not dissolved in the VDM solution. The VDM monomer wasdecanted and the grafted PMMA dissolved in chloroform and precipitatedinto hexane. This step was repeated two times to remove ungrafted VDMmonomer. Finally, the azlactone-functional PMMA support was dissolved inchloroform, casted onto an aluminum plate,and peeled away from the plateto form a thin film for analysis. Transmission IR spectrum of thegrafted PMMA showed there was a strong peak of carbonyl group of VDM at1820 cm⁻¹.

To prepare amine-terminated heparin, 1 g of heparin (commerciallyavailable from Diosynth) was dissolved in 300 mL of water. 10 mg SodiumNitrite was added to the solution and adjusted the pH to 2.7 with 1Nhydrochloric acid. The solution was adjusted to 7.0 and dialyzed against3 L of water in 3500 molecular weight cut-off dialysis tubing. Thesolution was concentrated and lyophilized to produce heparin-aldehyde.

1 g of heparin-aldehyde was dissolved in 100 mL of the buffer solution(1% citrate, 0.9% sodium chloride, pH=6.5). 0.5 g of ammonium sulfateand 0.25 g of sodium cyanoborohydride were added to the solution andstirred for five hours at room temperature. The solution was dialyzedwith 3500 molecular weight cut-off dialysis tubing against water. Thesolution was concentrated and lyophilized to produce amine-terminatedheparin.

Then, to react amine-terminated heparin with azlactone-functional PMMA,the film prepared according to this example was reacted with a solutionof 0.25 g of amine-terminated heparin which was dissolved in 50 mL ofbuffer solution (pH=8.8), and stirred for several hours at roomtemperature. The heparinized-film was rinsed with water and dipped in 1%solution of toluidine blue (Sigma Chemical) in water for staining. Thefilm stained a violet color within a few minutes to show heparin wasattached to the film.

EXAMPLE 54 Azlactone-functional PMMA Prepared in Heptane

PMMA casted film (Perspex C.Q./uv from ICI; less than 0.1 mm thickness)and a PMMA button (2.75 mm thickness) were both exposed to electron beam(10 kGys at 120 kV) in a N₂ blanket and then conveyed into the glove boxunder N₂ gas where the activated film and the activated button were bothimmersed in a mixture of heptane/VDM (75/25 wt. %) at room temperatureand 45° up to 15 hours. After grafting, both forms of the VDM-graftedPMMA were washed with anhydrous ethyl-ether. ATR-FTIR spectra of theVDM-grafted PMMA showed there was a strong peak of carbonyl group of VDMat 1820 cm⁻¹.

EXAMPLE 55 Azlactone-functional PMMA Prepared with Corona Discharge

The corona-discharge assembly of Example 17 above was placed in a glovebox filled with N₂ gas. PMMA films (from ICI and less than 0.1 mmthickness) were exposed to corona-discharge in different conditions at150 to 300 watts for 0.4-4 seconds exposure discharged at 62 kHz (at 150watts for 0.4 and 4.0 secs.; at 200 watts for 1.2 and 4.0 secs.; and at300 watts for 1.2 and 4.0 secs. The activated PMMA films were immersedin heptane/VDM mixture (75/25 wt. %) at room temperature for 2 hours.Evidence of VDM grafting was observed by ATR-FTIR. In these instances,there was no evidence of molecular weight degradation. From studyingthese examples 52-55 and the prior examples forming biologically activeand useful adduct supports, it is possible to convert a hydrophobic PMMAuseful as an intraocular lens into a hydrophilic PMMA coated with ananticoagulant or other biocompatible hydrophilic and/or biologicallyactive material by reaction of azlactone-functional moieties with ananticoagulant or another nucleophile-terminated hydrophilic moiety.

EXAMPLE 56 Dispersion Polymerization of Azlactone-Functional Particlesin Polyethylene Membranes

Three microporous PE membranes prepared according to Example 23 of U.S.Pat. No. 4,539,256 (Shipman) and one hydrophilized microporous PEmembrane prepared according to Example 22 of copending, coassignedapplication Ser. No. 07/775,969 (Gagnon et al.), each having a size of15 cm×15 cm, were each placed on a slightly larger piece of dense PEfilm and were saturated with a solution containing 10 g of VDM; 10 g ofethylene glycol dimethacrylate (EGDMA); 30 g of2-hydroxyethylmethacrylate (HEMA); 1 g of photoinitiator (Darocure 1173commercially available from E Merck), and 400 ml of isopropyl alcohol(net solids of 11.1 wt. %). Another piece of dense PE film was thenplaced on top of the saturated membrane followed by rubbing the"sandwich" construction to remove any excess solution. The sandwichconstruction was place in the bottom of a N₂ purged box having a Pyrexbrand glass top, and the sandwich construction was irradiated throughthe Pyrex window using two fluorescent UV "black" lights havingemissions at a maximum of 360 nm. The three PE samples were irradiatedfor 5 min, 10 min, and 15 min, respectively. The hydrophilized PE samplewas irradiated for 10 min. Following irradiation, the sandwichconstructions were removed from the box, separated from the PE filmsheets. Except for the PE sample irradiated for 10 min, which wasdirectly rinsed with isopropyl alcohol, each sample was dried and thenrinsed with isopropyl alcohol for 10 min. Scanning electron microscopy(SEM) of the surface and cross-section of each sample was performed.Except for the hydrophilized PE sample, SEM observation ofcross-sections showed numerous particles and particle clusters.Individual particles were seen between 0.4 and 0.8 μm in diameter.However, many particles were also agglomerated into a mass which waslarge as 5 μm in diameter. On the surface of these samples was a 2-5 μmthick cake of agglomerated particles having average diameters of about0.6 μm, and on the surface of all samples but the PE sample rinsedbefore drying, there were areas of a dense skin layer of about 1-2 μmthick on the outside surface. The PE sample rinsed directly wasskin-free and had many particles trapped within the void spacesthroughout the membrane cross-section. The hydrophilized PE sample wasmostly skinned and contained few if any particles within thecross-section. The interior of this membrane appeared to have a roughcoating on the fibril surfaces. Infrared spectroscopy of the PE samplerinsed directly showed strong absorbances at 1822 cm⁻¹ and 1671 cm⁻¹(indicating VDM) and at 1728 cm⁻¹ (HEMA and EGDMA) which confirmed thepresence of crosslinked VDM/HEMA copolymer.

EXAMPLE 57 Dispersion Polymerization of Azlactone-functional Particlesin Polypropylene Membrane

A microporous PP membrane (prepared according to Example 9 of U.S. Pat.No. 4,726,989 (Mrozinski)) was treated in the same manner as thehydrophilized PE membrane in Example 55. In this instance, the samplewas skin-free and had many particles trapped within the void spacesthroughout the membrane cross-section.

EXAMPLE 57 Dispersion Polymerization of Azlactone-functional Particleswith a Stabilizer

The procedure of Example 55 was repeated with the addition of 2.23wt./vol % of poly(vinyl pyrrolidone) (PVP K-30 commercially availablefrom EM Sciences) to the monomer mixture of Example 55 and saturatedinto the pores of a PE membrane prepared according to Example 23 of4,539,256 (Shipman). After irradiation, isopropyl alcohol rinsing wasemployed. For comparison, the same PE membrane was saturated with themixture of Example 55. Without PVP stabilizer, a bimodal distribution ofparticles measuring about 0.15 μm in diameter were found within thepores of the PE membrane with a few 2.0 μm diameter particles sparselyscattered through the sample. With the PVP present in the monomermixture, the particles were about 0.2-0.5 μm with no occurrence of themuch larger particles. Thus, PVP stabilizer improves the particle sizedistribution particles within void spaces of the PE membrane.

EXAMPLES 58-65 Dispersion Polymerization without Alteration of theSupport Beyond Usefulness

PP membranes samples prepared according to Example 9 of U.S. Pat. No.4,729,989 (Mrozinski) were used as the outer layers in a three layermembrane stack, having a middle layer of a hydrophilized PE membraneprepared according to Example 23 of U.S. Pat. No. 4,539,256 (Shipman)and hydrophilized according to Example 22 of copending, coassignedapplication Ser. No. 07/775,969 (Gagnon et al.) The hydrophilized middlelayer is denominated the H-PE layer. The membrane stack was placed upona piece of dense LDPE film and saturated with a monomer solution of 0.79moles VDM, 0.48 moles EDGMA, 0.25 moles HEMA, containing 2% Darocure1173 photoinitiator, dissolved in isopropyl alcohol in the amount ofpercent solids shown in Tables 17 and 18 below, according to theirtheoretical percent solids. Examples 58-61 were the top layer of PP ineach sample; Examples 62-65 were the middle layer, H-PE, in each sample.(Unmodified controls were also tested.)

After saturation, another piece of dense LDPE film was placed to coverthe membrane stack,and the excess solution was squeezed out with arubber roller. The membrane stack and the cover LDPE films were thenirradiated for 20 minutes with a bank of 4 fluorescent "black lights"having an emission maximum at 360 nm, under ambient temperature,pressure, and atmosphere.

Samples of Examples 58-65 were subjected to BET analysis (using themethod described in Example 3 above), and pore size analysis, Gurleyanalysis, porosity, and water permeability analysis (using the methodsdescribed in PCT Publication WO 92/07899 (Gagnon et al.)). The resultsfor surface area are shown in Table 17; the porous property results areshown in Table 18. Examples 58-61 show that where distinct dispersionbeads develop within the pores of the membrane, the surface area issignificantly enhanced. This enhancement is surprisingly obtainedwithout a significant decrease of other microporous properties of themembranes, as seen in Table 18.

By contrast, the results of Examples 62-65 show that a coating ofcrosslinked VDM-co-HEMA is formed in situ on the internal pore surfaces.This is shown by a decrease in specific area/frontal surface area ratio,with the exception being Example 65, which had a "lacy" network ofcrosslinked dispersion beads filling the pores of the middle layer H-PE.Also in contrast to particulate additions, coatings of Examples 62-64served to decrease somewhat the flow through properties (see Gurley andwater permeability properties) of the membrane, although the pore size,thickness, and porosity values were not similarly affected beyondusefulness of the azlactone-functional support.

                                      TABLE 17                                    __________________________________________________________________________    SURFACE AREA OF DISPERSION POLYMERIZED SAMPLES                                        Monomer                Total  % Change                                        Solution                                                                            Area/Mass                                                                           Wt Add On                                                                           Basis Wt.                                                                          Area/Surface                                                                         in Area/                                Example (% Solids)                                                                          (m.sup.2 /gr)                                                                       (%)   (gr/m.sup.2)                                                                       (m.sup.2 /m.sup.2)                                                                   Surface                                 __________________________________________________________________________    PP Control                                                                            --    20.9  00.0  16.000                                                                             334.40 --                                      58      2.8   23.9  1.50  16.240                                                                             388.14 16.1                                    59      5.7   25.6  9.90  17.584                                                                             450.15 34.6                                    60      11.3  15.0  41.00 22.560                                                                             338.40 1.2                                     61      22.6  17.2  57.60 25.216                                                                             433.72 29.7                                    H-PE Control                                                                          --    18.4  0.00  14.700                                                                             270.48 --                                      62      2.8   17.2  0.90  14.832                                                                             255.12 -5.7                                    63      5.7   13.8  11.30 17.920                                                                             247.30 -8.6                                    64      11.3   9.8  25.80 21.800                                                                             213.64 -21.0                                   65      22.6  10.5  44.00 32.700                                                                             343.35 26.9                                    __________________________________________________________________________

                                      TABLE 18                                    __________________________________________________________________________    POROUS PROPERTIES OF DISPERSION POLYMERIZED SAMPLES                                                               Water                                             Monomer                     Permeability                                      Solution                                                                            Thickness                                                                           Pore Size                                                                          Gurley Wt.                                                                          Porosity                                                                           (ml/min/cm.sup.2)                         Example (% Solids)                                                                          (μm)                                                                             (μm)                                                                            (sec/50 cc)                                                                         (%)  Area/Surface                              __________________________________________________________________________    PP Control                                                                            0.0   51    0.47 10    77.6 6.34                                      58      2.8   53    0.56 11    73.6 8.66                                      59      5.7   58    0.51 12    74.0 7.43                                      60      11.3  58    0.51 16    66.1 4.93                                      61      22.6  91    0.49 14    75.5 4.82                                      H-PE Control                                                                          0.0   61    0.47 33    71.4 3.68                                      62      2.8   53    0.62 21    69.7 --                                        63      5.7   56    0.57 62    61.8 1.46                                      64      11.3  69    --   >300  66.8 --                                        65      22.6  61    0.47 >300  66.4 <0.70                                     __________________________________________________________________________

Examples 63-65 were also subjected to testing for protein couplinganalysis. The samples were tested using radioactively labeled protein A,according to the procedures of Examples 5, 6 and 10 above by incubatingovernight in radiolabeled Protein A, buffered with either 1.5M sulfatebuffer (SO₄) or 250 mM phophate buffered saline (PBS), both at pH 7.5.After quenching of unreacted azlactone moieties with ethanolamine andrepeated rinsing in buffer, scintillation counting was done to determineinitial binding levels. The samples were then incubated for 4 hours in1.0% sodium dodecylsulfate (SDS), followed by scintillation counting todetermine the amount of coupled protein remaining. Table 19 shows theresults.

                                      TABLE 19                                    __________________________________________________________________________    PROTEIN BINDING PROPERTIES OF DISPERSION POLYMERIZED SAMPLES                                            Protein    Protein                                           Wt. Add On                                                                           Area/Surf*                                                                          Buffer                                                                            Binding                                                                            SDS Res.                                                                            Coupling                                 Example  (%)    (m.sup.2 /m.sup.2)                                                                  Type                                                                              (μg/cm.sup.2)                                                                   (%)   (μg/cm.sup.2)                         __________________________________________________________________________    PP Control                                                                              0.00  270.48                                                                              SO.sub.4                                                                          21.1 27.6  5.8                                      63       11.30  247.30                                                                              SO.sub.4                                                                          38.1 79.6  30.3                                     64       25.80  231.64                                                                              SO.sub.4                                                                          16.7 86.0  14.2                                     65       44.00  343.35                                                                              SO.sub.4                                                                          48.6 87.6  42.3                                     H-PE Control                                                                            0.00  270.48                                                                              PBS 21.2 30.6  6.5                                      63       11.30  247.30                                                                              PBS 15.1 63.0  9.6                                      64       25.80  213.64                                                                              PBS  6.7 58.8  4.0                                      65       44.00  343.35                                                                              PBS  5.5 60.2  3.3                                      __________________________________________________________________________     *See Table 17 above.                                                     

These data show that the addition of azlactone functionality doescorrelate with an increase in the percent of coupled protein, and thatuse of sulfate is preferred to use of saline as a buffer system.

The experiment was repeated for samples of Example 63, except that theexperiments were done as a function of the time allowed for initialbinding. The amount of time ranged from 0.5 hours to 16 hours andresulted in initial binding ranging from 21 μg/cm² for 0.5 hours to 48.9μg/cm² for 16 hours.

The experiment was then repeated for samples of Example 63, except thatrather than incubating in the protein A solution, the protein A solutionwas flowed through the azlactone-functional membrane. In this case, 3 mlof a 1 mg/ml solution of non-radioactive, SO₄ -buffered protein Asolution at pH=7.5 was suctioned through a 25 mm disk of theazlactone-functional membrane using aspirator vacuum. After threeflow-through rinse cycles with PBS buffer solution and quenching ofpossible unreacted azlactone moieties with 6 ml of 1M ethanolamine(buffered to pH=9 with 25 mM pyrophosphate), the membrane samples wereanalyzed for Protein A content using the BCA protocol published byPierce Chemical Co. for BCA Protein Assay Reagent (Cat. No. #3220/23225,Pierce Chemical Co., Rockford, Ill.). It was found that the membranesinitially bound an average of 22.3 μg/ml of protein A using theflow-through mode of binding, where the time of exposure to the proteinsolution was less than about 3 minutes. This amount of initial bindingwas consistent with initial binding levels for Example 63 using a 0.5hour incubation. The advantage of flow-through binding is that bindingis not limited by the rate of diffusion of protein into pores of themembrane. Flow-through binding also demonstrated that kinetics ofinitial binding of protein to azlactone moieties is apparently veryrapid.

EXAMPLE 66-69 Retained Useful Porous Properties of Azlactone-functionalSupports

Membranes were prepared according to Example 3 above, using 50 kGyradiation and 10, 15, or 20 wt/vol % VDM solutions as listed in Table 20below. Although a significant amount of poly(VDM) was grafted to themembranes, it is apparent from measurements of physical properties thatno significant change in the physical porous properties occurred. Mostimportantly, flow properties were not diminished beyond continuedusefulness of the azlactone-functional membranes.

                                      TABLE 20                                    __________________________________________________________________________    POROUS PROPERTIES OF DISPERSION POLYMERIZED SAMPLES                                                     Gurley   Water                                                             Pore                                                                             Wt.      Permability                                     VDM Conc.                                                                            Wt. %                                                                              Thickness                                                                           Size                                                                             (sec/50                                                                           Porosity                                                                           (ml/mn/cm.sup.2                            Example                                                                            (wt/vol %)                                                                           Add-On                                                                             (μm)                                                                             (μm)                                                                          cc) (%)  @ 10 psi)                                  __________________________________________________________________________    66    0      0.0 51    0.54                                                                             22  67.5 3.68                                       67   10     10.1 52    0.59                                                                             21  65.9 2.91                                       68   15     13.3 51    0.56                                                                             23  66.3 3.12                                       69   20     15.2 47    0.55                                                                             22  66.2 1.60                                       __________________________________________________________________________

The samples of Examples 66-69 were also tested for ability to coupleprotein A according to the method of Example 5 above. The protein wasdissolved in either a SO₄ or a PBS buffer system as used in Examples63-65. Table 21 shows the results below.

                                      TABLE 21                                    __________________________________________________________________________    PROTEIN BINDING PROPERTIES OF GRAFTED SAMPLES                                                        Protein    Protein                                           IR Ratio*                                                                           Wt Add On                                                                            Buffer                                                                            Binding                                                                             SDS Res.                                                                           Coupling                                    Example                                                                             (wt/vol 5)                                                                          (%)    Type                                                                              (μg/cm.sup.2)                                                                    (%)  (μg/cm.sup.2)                            __________________________________________________________________________    66    0.000 0.0    SO.sub.4                                                                          18.3  31.4 5.7                                         67    0.543 10.1   SO.sub.4                                                                          33.2  94.3 31.3                                        68    0.691 13.3   SO.sub.4                                                                          29.5  94.8 27.8                                        69    0.662 15.2   SO.sub.4                                                                          32.6  94.1 30.6                                        66    0.000 0.0    PBS 17.8  29.9 4.3                                         67    0.543 10.1   PBS 8.9   84.4 7.6                                         68    0.691 13.3   PBS 8.1   75.9 6.1                                         69    0.662 15.2   PBS 6.3   85.1 5.4                                         __________________________________________________________________________     *Ratio of the azlactone absorbance of 1824 cm.sup.-1 to that of the PE at     1462 cm.sup.-1                                                           

These data show that the addition of azlactone functionality doescorrelate with an increase in coupled protein. Also, the SO₄ buffersystem was preferred.

Samples of Example 67 were also tested for rate of binding duringincubation binding and flow-through binding techniques in the samemanner as used for Example 63 above. For the incubation bindingtechnique, the amount of initial binding ranged from 21.8 μg/cm² for 0.5hours incubation to 37.4 μg/cm² for 16 hours of incubation. For theflow-through binding technique, the amount of initial binding was anaverage of 20.4 μg/ml for a flow-through exposure of about 3 minutes. Aswith Example 63, flow-through binding techniques are preferred and alsodemonstrate continued usefulness of the pre-existing support afterazlactone-functionality is added thereto.

Embodiments of the invention are not limited by the above descriptionand examples. For an appreciation of the scope of the invention, theclaims follow.

What is claimed is:
 1. An adduct support, comprising: a chemicallyreactive support having azlactone-functionality at surfaces of thesupport and a ligand comprising a nucleophilic reagent reacted with theazlactone-functionality, wherein the chemically reactive supportcomprises a porous pre-existing support having surfaces andazlactone-functional moieties contacting only the surfaces and modifyingreactivity of only such surfaces while retaining useful porosity of thepre-existing support;wherein said contacting is selected from the groupconsisting of chemically grafting the azlactone-functional moieties tothe surfaces, crosslinking the azlactone-functional moieties in acoating over the surfaces, and forming crosslinked particles of theazlactone-functional moieties in contact with the surfaces.
 2. Theadduct support according to claim 1, wherein the azlactone-functionalmoieties comprise oxazolinone moieties of the formula: ##STR4## whereinR¹ and R² independently can be an alkyl group having 1 to 14 carbonatoms, a cycloalkyl group having 3 to 14 carbon atoms, an aryl grouphaving 5 to 12 ring atoms, an arenyl group having 6 to 26 carbon atomsand 0 to 3 S, N, and nonperoxidic O heteroatoms, or R¹ and R² takentogether with the carbon to which they are joined can form a carbocyclicring containing 4 to 12 ring atoms, andn is an integer 0 or
 1. 3. Theadduct support according to claim 1, wherein the pre-existing support isa ceramic, glassy, metallic, or polymeric material.
 4. The adductsupport according to claim 3, wherein the pre-existing support is apolymeric material.
 5. The adduct support according to claim 4, whereinthe porous, polymeric material is a woven web, a nonwoven web, amicroporous fiber, or a microporous membrane.
 6. The adduct supportaccording to claim 1, wherein the nucleophilic reagent comprisesbiologically active materials, acids, bases, chelators, hydrophiles,lipophiles, hydrophobes, zwitterions, detergents, or combinationsthereof.
 7. The adduct support according to claim 6, whereinbiologically active material comprises substances which arebiologically, immunochemically, physiologically, or pharmaceuticallyactive.
 8. The adduct support according to claim 7, wherein thebiologically active material comprises proteins, peptides, polypeptides,antibodies, antigenic substances, enzymes, cofactors, inhibitors,lectins, hormones, receptors, coagulation factors, amino acids,histones, vitamins, drugs, cell surface markers, substances whichinteract with them, or combinations thereof.
 9. The adduct supportaccording to claim 6, wherein the adduct support is an adsorbant,complexing agent, catalyst, or chromatographic article.
 10. The adductsupport according to claim 6, wherein the adduct support is a mammalianbody implant and wherein the nucleophilic reagent is an anticoagulant.